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Full text of "Report of the President's Biomedical Research Panel : Supp 1"

NEW BOOK RACK 




Report of the 

President's 
Biomedical Research 

Panel 



SUPPLEMENT 1 
Analysis of Selected Biomedical Research Programs: 

Case Histories 



S^^/ ^^si/^-'j ^- y'^^^s.^o^ /^^ ^ y-^</77 



^port of the 

President's 
Biomedical Research 

Panel 



SUPPLEMENT 1 
Analysis of Selected Biomedical Research Programs: 

Case Histories 

Battelle-Columbus Laboratories 
(Contract Number NOl-PP-6-2104) 



April 30, 1976 



U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE 
LJ.I Public Health Service 
DHEW Publication No. (OS) 76-506 



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For sale by the Superintendent of Documents, U.S. Government Printing Office 
Washington, D.C. 20402 - Price $3.40 

Stoctc No. 040-000-00351-7 



Preface 



The President's Biomedical Research Panel was established on January 29, 1975, and charged, 
under Public Law 93-352, to review and assess the conduct, support, policies, and management of 
biomedical and behavioral research as conducted and supported through programs of the National 
Institutes of Health (NIH) and the Alcohol, Drug Abuse, and Mental Health Administration 
(ADAMHA). The legislation directs the Panel to submit a report of its findings by April 30, 1976, 
to the President and to the Congress. 

Over the period of fifteen months, the seven members of the Panel conducted an extensive 
study that involved assessments of the state of the science, the impact of federally funded re- 
search on institutions of higher education, the organization and management of the NIH and the 
ADAMHA, the dissemination and appUcation of research findings, and the development of policy 
for federal support of biomedical and behavioral research. 

The main volume of this report (DHEW Publication No. (OS)76-500) contains the issues iden- 
tified by the Panel in its study and the Panel's subsequent recommendations. The annex included 
in this volume details the methods of study used by the Panel and provides hstings of witnesses 
who appeared before the Panel in hearings, professional and volunteer organizations contacted 
for submission of views, and resource materials available to the Panel in the course of its delibera- 
tions. Four appendices and four supplementary volumes accompany the main report. 

Appendix A, "The Place of Biomedical Science in Medicine and the State of the Science" 
(DHEW Publication No. (OS)76-501), contains the reports of the Overview and Interdisciplinary 
Clusters. In an unprecedented study, the Panel brought together preeminent biomedical and 
behavioral scientists and asked them, on an interdisciphnary basis, to assess the state of the 
science, to identify areas of promise and the resources required to achieve important new knowl- 
edge, and to relate research to the health of the nation's people. The results of this effort are 
contained within the reports of the eleven Interdisciplinary Clusters. The report of the Overview 
Cluster focuses on the place of biomedical science in medicine. 

Appendix B, "Approaches to Policy Development for Biomedical Research: Strategy for 
Budgeting and Movement from Invention to Chnical AppUcation" (DHEW Publication No. 
(OS)76-502), presents the results of three studies performed under contract to the Panel: 
(1) "Lags Between Initial Discovery and Chnical Application to Cardiovascular Pulmonary Medi- 
cine and Surgery" (Julius H. Comroe, Jr.), (2) "Analysis of Selected Biomedical Research 
Programs" (BatteUe-Columbus Laboratories), and (3) "Policy .Analysis for Federal Biomedical 
Research" (The Rand Corporation). 



Ill 



Appendix C, "Impact of Federal Health-Related Research Expenditures Upon Institutions of 
Higher Education" (DHEW Publication No. (OS)76-503), presents a summary of findings and 
conclusions of studies on the influence of federal funding of biomedical and behavioral research 
upon the financial status and educational functions of research universities and academic medical 
centers. The studies were conducted, under contract, by the American Council on Education, the 
Association of American Medical Colleges, and The Rand Corporation. 

Appendix D, "Selected Staff Papers" (DHEW Publication No. (OS)76-504), offers eight papers 
prepared by individual members of the Panel staff on a variety of topics pertinent to the NIH 
and the ADAMHA. 

All five of the above mentioned volumes are available from the Government Printing Office, 
as indicated. 

In addition, four volumes of supplementary resource documents are also available from the 
Government Printing Office. 

Supplement 1. "Analysis of Selected Biomedical Research Programs: Case Histories" (DHEW 
Publication No. (OS)76-506). Background information for the study by 
Battelle-Columbus Laboratories. 

Supplement 2. "Impact of Federal Health-Related Research Expenditures Upon Institutions 
of Higher Education" (DHEW Publication No. (OS)76-507). Findings, con- 
clusions, and background information of studies by the American Council on 
Education, the Association of American Medical Colleges, and The Rand 
Corporation. 

Supplement 3. "Written Statements Supplementing Verbal Testimonies of Witnesses" (DHEW 
Publication No. (OS)76-508). 

Supplement 4. "Statements of Professional, Scientific, and Voluntary Health Organizations" 
(DHEW Publication No. (OS)76-509). 

Verbatim transcripts for all meetings of the Panel, except the March 1975 meetmg, are avail- 
able from the U.S. Department of Commerce, National Technical Information Service, 
Springfield, Virginia 22161. 



IV 



TABLE OF CONTENTS 
VOLUME II. APPENDIXES 

A. FENESTRATION 

B. VENTRICULAR SEPTAL DEFECT 

C. KIDNEY TRANSPLANTATION 

D. ORAL CONTRACEPTIVES 

E. PENICILLIN 

F. L-DOPA 

G. PHENOTHIAZINES 
H. CHLOROTHIAZIDE 

I. COMBINATION CHEMOTHERAPY OF ACUTE CHILDHOOD LEUKEMIA 

J. THE CARDIAC PACEMAKER 

K. EMI SCANNER 

L, POLIO VACCINE 

M. RH ANTISERUM ("VACCINE") 

N. RUBELLA VACCINE 

O. PHENYLKETONURIA 

P. AUSTRALIA ANTIGEN 

Q. GLUCOSE -6 -PHOSPHATE DEHYDROGENASE DEFICIENCY 

R. ALPHA-FETOPROTEIN 

S. PRIOR STUDIES OF RESEARCH AND INNOVATION 

T. GLOSSARY 



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A-1 



APPENDIX A. FENESTRATION 



INTRODUCTION AND SUMMARY 

A National Health Survey has indicated that 14 million people in the United States 
are afflicted with defective hearing (Schein, 1974). Although hearing loss has been 
ascribed primarily to the elderly, this is a misconception since almost three out 
of five victims are under age 65 (Lubart, 1963). 

Deafness is divided by otologists into two groups, conductive and sensorineural 
deafness. Conductive deafness includes any hearing loss associated with the trans- 
mission of sound from the environment to the cochlea, while nerve or sensorineural 
deafness involves hearing loss associated with the cochlea or higher auditory pathways. 
Of these two types of deafness, only the conductive type has been satisfactorily treated 
via surgical procedures, especially those surgical procedures involving otosclerosis. 

Estimates of the number of people in the United States suffering from otosclerosis 
vary, but is is safe to say that over 2 million people are afflicted (Valvassori, 1965). 
Otosclerosis has been demonstrated in homo sapiens only and is found most often in 
Caucasians, and only occasionally in Negroid or Mongoloid man. Although otosclerosis 
is encountered almost twice as frequently in females as in males during clinical 
practice, this does not reflect the true sex ratio. This is because unilateral deafness 
is much more common in men, and as a rule, a patient with unilateral deafness is less 
likely to seek medical advice. Thus, otosclerosis is only slightly more common in 
womien than in men. 

Otosclerosis is a hereditary and chronic disease that causes hearing loss of the 
conductive type. It occurs in certain regions of the temporal bone and produces symp- 
toms by interfering with the function of the middle ear ossicular chain or in the inner 
ear nerve cells, or both. The commonest site of origin of otosclerosis is in front of 
the oval window, but the disease can also arise in the footplate of the stapes itself, in 
the region of the round window, or in other parts of the cochlear capsule. 

There are three principal types of otosclerosis: (l) histological - an otosclerotic 
focus which causes no symptoms but can be detected at necropsy; (2) clinical or stape- 
dial - the most commonly encountered form in which the otosclerotic focus progressively 
affects the footplate of the stapes via increasing fixation (ankylosis); and (3) cochlear — 
otosclerotic foci which affect the endosteal bone surrounding the cochlea or labyrinth 
by producing secondary sensorineural changes (Morrison, 1970). 

The term "otosclerosis" as applied by von Troltsch in 1881 was inaccurate because 
he thought that fixation of the stapes was produced by sclerosing changes in the tympanic 
mucosa (as cited in Valvassori). In 1893, Politzer recognized that stapes fixation was 
due to a disease of the labyrinthine capsule, and in 1912, Siebenmann proposed the term 
"otospongiosis" because the affected bone was less dense than the normal otic capsule. 
However, the term otosclerosis still persists and is the generally accepted term. As 
it stands, otosclerosis refers to all cases of primary labyrinthine capsule disease, 
with or without fixation of the stapes. 

At the present time, surgery is the only proven treatment for patients with 
otosclerosis that is uncomplicated by cochlea involvement and sensory- neural loss. 



A- 2 

Three basic types of surgical procedures have evolved for treating otosclerosis: 
(1) fenestration, (2) stapes mobilization, and (3) stapedectomy. Fenestration of the 
horizontal semicircular canal is based on the principle of establishing a new sound 
pathway in place of the occluded oval window. Stapes mobilization involves freeing 
the ankylosed or fixed stapes by to- and- fro palpation, and stapedectomy, which may 
be partial or total, consists of removing the stapes and replacing it with a mobile 
prosthesis. 

The first approach to the surgical treatment of otosclerosis occurred a century 
ago when Kessel in 1876 mobilized and 3 years later extracted the stapes. Towards the 
end of the nineteenth century, pioneers such as Boucheron, Miot, and Jack were per- 
forming transtympanic mobilization or extraction of the stapes. However, without 
adequate illumination, magnification, or antibiotics, most of these patients died 
because of postoperative labyrinthitis and meningitis. .As a result, these operations 
fell into disrepute and •were abandoned. 

Later, in 1913, Jenkins described a method for bypassing the middle ear by 
means of an opening made into the lateral semicircular canal. This stimulated further 
investigation on modifications of the procedure in an attempt to prevent closure of the 
fenestra and onset of infection. The first definitive practical approach to the surgery of 
otosclerosis occurred in 1938 ■when Lempert described his one- stage fenestration 
operation. This operation flourished from approximately 1938 to 1955 and represented 
the first major breakthrough in surgery for deafness. 

Lempert's one- stage technique involved exposing and removing the mastoid 
cells through endaural incisions. After the incus and head of malleus had been removed 
to provide better exposure, a fenestra was made in the most accessible portion of the 
horizontal canal and covered with a thin membrane obtained from the canal wall. Although 
this operation was a great step for'ward, the fenestra would sometimes close after a 
variable length of time. In addition, the large mastoid cavity and open ear canal 
thus created would drain for long periods of time. As a result, weeks of hospitalization 
and months of postoperative care were required. 

Despite its drawbacks, the fenestration operation was the established treatment 
until the early 1950's when Rosen devised a procedure known as stapes mobilization. 
Stapes mobilization had the advantage of requiring only 1 or 2 days of hospitalization 
and very little postoperative care. Rosen's technique provided impetus for a ne'w 
generation of otologists ■who ■were no^w benefiting from microsurgery techniques, better 
lighting, and antibiotics, and ■who ■were well trained in both fenestration and mobilization 
procedures. As a result, it ■was inevitable that interest should be rea^wakened in the 
possibilities of removing the ankylosed stapes. 

In 1958, Shea introduced his operation which involved replacing the stapes by 
a vein graft and polyethylene strut prosthesis. Stapedectomy in one form or another 
is the basic surgical treatment for otosclerosis today and the classic Lempert fenes- 
tration operation has been virtually discarded. 



A- 3 
HISTORICAL ACCOUNT 



The historical account is divided into four sections: (I) description and etiology 
of otosclerosis, (2) diagnostic procedures, (3) surgical procedures, and (4) contributing 
events. For each section, key chronological events are discussed, although sonie of the 
events are not presented on the historiograph nor are all events shown on the historio- 
graph discussed. The historiograph illustrates the interrelationship of events from a 
chronological view. 

Description and Etiology of Otosclerosis 

The development of otology and especially the study of the ear as a structure dates 
back to 500 B, C. when Alcm.aeon discovered the otopharyngeal passage (eustachian tube) 
(as cited in Saunders, 1971). * Hippocrates in 400 B. C. was the first to inspect the tym- 
panic membrane and recognize it as a part of the organ of hearing. In addition, he clearly 
described acute otitis. However, the history of basic ear research events begins with 
Duverney in 1683. Duverney is called the "father of otology" because as a physiologist, 
anatomist, and pathologist, he made many important contributions to understanding the 
ear as a hearing organ. These included: 

(1) Correcting the belief that the eustachian tube served as a breathing or hearing 
mechanism, and stated that it was simply a channel through which tympanic air 
■was renewed 

(2) Publishing the first monograph on otology 

(3) Demonstrating that sound could be transmitted via air in the middle ear 

(4) Suggesting first that different pitches were perceived in different areas of the 
cochlea. 

In 1704, Valsalva related deafness to stapedial ankylosis and described the 
anatomy of the ear as seen in over 1000 temporal-bone dissections. During the next 
150 years, various structures or components of the ear were investigated. In 1853, 
Wilde wrote the medical classic, "Practical Observations on Aural Surgery and the 
Nature and Treatment of Diseases of the Ear". As a result, he is known as the 
"father of modern otology" because of the following. 

(1) He developed "Wilde's incision", an incision of the mastoid down through the 
periosteum for fluctuant mastoiditis. 

(2) He invented the angled shaft on aural instruments. 

(3) He publicized ear diseases. 

In 1857, Toynbee was the first to correlate ear pathology with clinical history by 
showing that osseous ankylosis of the stapes to the oval window is one of the commonest 
causes of deafness. Meniere, in 1861, established that vertigo is an affection of the 
internal ear and accurately described the symptom complex which bears his name. 

"Many of the early events have been described by Saunders (1971). 



A- 4 

Helmholtz in 1863 published a treatise on middle- ear functions and in 1869 des- 
cribed the transformer mechanism of the middle-ear structures, including the lever 
action of the drumhead and ossicles and the hydraulic action of the large tympanic 
membrane acting on the small stapedial footplate. 

The term "otosclerosis" wa.s first applied to stapes ankylosis by von Troltsch in 
1881 in reference to sclerosing changes in the tympanic membrane which he believed 
was causing fixation of the stapes. In 1893, Politzer described the histologic findings 
associated with stapes fixation and was the first to associate stapes ankylosis as a 
primary disease of the labyrinthine capsule. It was his findings that led Siebenmann in 
1912 to propose the term "otospongiosis" since the pathologic bone was more porous and 
less dense than the normal capsular bone it replaced. However, the otosclerosis term 
had come into such general use that Siebenmann's term, although more accurate, never 
materialized. 

In 1932, von Bekesy identified the mechanisms of excitation due to air and bone 
conduction stimulation. Although he confirmed Helmholtz's localization of stimuli within 
the cochlea, he changed the resonance theory as proposed by Helmholtz. He was able to 
show that the special hydrodynamic conditions prevailing in the cochlea permiitted move- 
mients of the footplate to set up a traveling wave complex in the basement memibrane, 
the highest crest of which occurs in the base with high pitches and toward the apex for 
low ones. As a result of his research efforts, von Bekesy was awarded the Nobel Prize 
in 1961. 

Diagnostic Procedures 



The first notable event in tests of auditory function occurred in 1711 when Shore 
invented the tuning fork. The various tuning-fork tests, i. e. , Weber (1834), Rinne (1855), 
and Schwabach (1885), are still in use all over the world, and are a very reliable method 
of determining the kind of deafness, conductive or sensorineural, that may be present. 
Innpaired function of the external or middle ear gives rise to conductive deafness, 
whereas impaired function of the inner ear or its central connection leads to sensor- 
ineural deafness. 

The next inraportant event occurred in 1878 when Hartmann devised the first 
audiometer, an electronic instrument that could deliver pure tones to the ear at any set 
frequency or intensity. However, the first con:imercial audiometer, as developed by 
Fletcher, did not appear until 1920. 

Significant contributions in this field were n^ade by Barany in 1906 when he 
invented a chair for use during vestibular testing. This test involved placing the patient 
in a rotating chair and, because during rotation the eyes exhibit nystagmus, an estimate 
of the response of the semicircular canals to stimulation was provided. In addition, 
Barany in 1914 received the Nobel Prize for his clarification of the physiology and 
pathology of the vestibular apparatus. 

In 1950, Carhart drew attention to a notch (depression) seen in audiometric tests 
on patients possessing an otosclerotic stapedial fixation. This notch (Carhart' s notch) 
observed at 2000 cps was due to stapedial fixation and was caused by the elimination of 
the middle- ear- bone conduction contribution. The alleviation of the notch observed 
after fenestration surgery was due to an improvement of the fluid inertial bone con- 
duction component of the inner ear. Thus, this test was indicative of stapedial fixation 
and served as a diagnostic aid to the otologist. 



A- 5 

Surgical Procedures 

The first significant event in ear surgery occurred in 1736 when Petit performed 
the first successful operation for mastoiditis. Further indications and techniques of 
the simple mastoid operation were described by Schwartze in 1873. 

A significant event in ear surgery occurred in 1876 when Kessel of Germany 
tried to mobilize the ankylosed stapes and, in a number of cases, actually extracted 
the stapes in the first application of stapedectomy. Stapes mobilization was also 
practiced by Boucheron (1888) and Miot (1890) in France. 

Stapes extraction for otosclerosis was first practiced in America by Blake 
(1892) and Jack (1892). However, both stapes mobilization and stapes extraction 
were condemned at this time by other otologists because the prevalence of post- 
operative infection made these operations dangerous. In addition, labyrinthitis and 
meningitis were ever-present risks if the protection of the stapedial footplate to the 
inner ear was weakened. During this same period, Kiister (1889) enumerated the 
steps upon which the radical mastoidectomy operation is based, but it was Zaufal 
(1890) who described Kilster's technique in detail, and as a result, his name is most 
often associated with that particular operation. 

In 1897, Passow reported the first operation for otosclerosis in which the 
ankylosed stapes was bypassed. An attempt was made to provide an alternative route 
for sound to enter the cochlea by drilling a hole into the promontory and covering this 
hole with a tympanic mucous membrane. Once again, this surgical procedure failed to 
arouse interest because of the possibility of ear infection. 

A decade passed before the next major stride in ear surgery occurred. This was 
accomplished by both Barany (1911) and Jenkins (1913) when they attempted to decom- 
press the labyrinth in otosclerosis (fenestration). Barany suggested a fistula into the 
posterior vertical canal, while Jenkins made an opening into the lateral semicircular 
canal in two patients. In one patient, he covered the fistula with a Thiersch graft and 
in the other case, he used a flap from the external meatus. Although temporary im- 
provement in hearing was noted in both instances, the procedure was abandoned when 
one patient went completely deaf and the other nearly so. 

In 1917 Holmgren revived interest again in the surgical correction of deafness 
by pioneering a two- stage fenestration operation. He performed a series of operations 
for otosclerosis and demonstrated that with sterile techniques the inner ear could be 
opened safely. In addition, in 1922, he introduced the operating binocular microscope 
to otosclerosis surgery. Finally in 1937, Sourdille of France restored hearing per- 
manently by means of fenestration for the first time in otosclerosis cases. His in- 
genious operation involved fenestrating the horizontal canal and covering it with a 
meatal skin flap. This multistage procedure was the basis upon which Lempert devel- 
oped the one-stage fenestration operation. 

The landmark event in the treatment of otosclerosis occurred in 1938 when 
Julius Lempert developed and reported a one- stage operation for fenestration. He 
made feasible the first practical surgical approach to the external semicircular 
canal and popularized the "fenestration operation" for otosclerosis. More importantly, 
it was this operation which encouraged other otologists to favor reconstructive surgery 
and, through his efforts, a renaissance in reconstructive surgery for conductive 
deafness occurred. 



A -6 

The Lempert "fenestration operation" became established throughout the world 
and, for many years, was the operation of choice for the relief of deafness caused by 
otosclerosis. However, the fenestration operation was largely replaced in the early 
1950's when Rosen (1952) accidentally rediscovered stapes mobilization. In spite of 
considerable opposition at first, he pioneered the development of direct surgery on the 
otosclerotic stapedial footplate. The technique of stapes mobilization was used during 
the period 1953-1958 but was eventually replaced by Shea's stapedectomy technique in 
1958. Stapedectomy is considered the beginning of the nnodern era of stapes surgery 
and, along with stapes mobilization, brought an end to the classic fenestration operation. 

Contributory Events 

The first significant event occurred in 1807 when Bozzini devised an aural specu- 
lum which contained mirror reflectors and illumination. This instrument made it 
possible to see the ear canal and tympanum, to observe any infection within the outer 
ear, and to note any changes in the middle ear as evidenced through the tympanic 
membranes. In 1841, Hoffman devised the first otologic head mirror for drumhead 
illumination. This was an indirect source of lighting, and in 1860, von Troltsch 
devised the first modern otoscope which provided for direct lighting of the ear. 

In 1864, Siegle introduced the pneumatic otoscope which permitted one to inflate 
and deflate a rubber bulb for testing the mobility of the tympanic membrane. It also 
permitted the location of any pinhole leaks in the membrane. 

A decisive event occurred in 1921 when Nylen introduced the monocular operat- 
ing microscope in ear surgery, and for the first time, the structures of the ear could 
be magnified. This permitted better observation and ease of structure manipulations 
during the course of surgery. 

Another decisive event occurred in 1929 with the initial development of antibiotics 
by Flemming. He showed that the mold Penicillium notatum produced a substance which 
was able to inhibit the growth of Staphylococcus aureus and yet was not toxic to animals. 

The potential of antibiotics did not begin to develop until the late 1930's and 
early 1940's. At this time, Domagk (1935) discovered the antibacterial properties of 
Prontosil in his experiments with azo dyes which later led to the sulfa drugs. In 
addition, he introduced the first compound with action of sulfanilamides agan'ist strep- 
tococcal infections (Prontosil dye). 

Finally in 1940 (Chain et al. ) and 1941 (Abraham et al. ), sufficient stable material 
from the penicillium mold was purified by Florey to perform certain tests and to initiate 
studies on human beings. This landmark development was to affect significantly the 
treatment of infection in otology and provide impetus for the surgical correction of 
conductive deafness. 



REFERENCES 



Abraham, E. P. , Chain, E. , Fletcher, CM., Florey, H. W. , Gardner, A. D. 
Heatley, N. G. , and Jennings, M. A. , Lancet, 2, 177 (1941). ' 



A-7 
Alderton, H. A., Tr. Am. Otol. Soc. , 7, 60 (1898). 
Bekesy, G. von, Ann. Physik, J^j ^ (1932). 

Bezold, F. , Textbook of Otology , Translated by J. Holinger, E. H. Colgrove Co., 
Chicago, 157 (1908). 

Blake, C. J., Tr. Am. Otol. Soc, 5, 306 (1892). 

Boucheron, Union med. , Par., 46_, 412 (1888). 

Carhart, R. , Arch. Otol., 5^, 798 (1950). 

Chain, E. , Florey, H. W. , Gardner, A. D. , Heatley, N. G. , Jennings, M. A. , 
Orr-Ewing, J., and Saunders, A. G. , Lancet, 2, 226 (1940). 

Fleming, A., Brit. J. Exp. Path., 10_, 226 (1929). 

Gelle, M. , Trans. Int. Med. Congr. , London, 3_, 7thSess., 370 (1881). 

Griessmann, B. , Beitr. Anat. Ohr. , ]_6_, 47 (1921). 

Guild, S. R., Ann. Otol. Rhin. & Laryng. , 53_, 246 (1944). 

Helmholtz, H. L. F. , Pfliigers Arch. ges. Physiol., j_, 1 (1968). 

Holmgren, G. , Acta Otolaryng, 5_, 460 (1923). 

Jack, F. L. , Tr. Am. Otol. Soc, 5, 474 (1893). 

Jenkins, G. J. , Tr. XVllth Internat. Congr. Med., London, 1_6, 609 (1913). 

Kessel, J., Arch. f. Ohrenh. , \3_, 69 (1878). 

Kiister, E. , Deutsche Med. Wchnschr. , _1_5, 254 (1889). 

Lempert, J., Arch. Otolaryng., 28, 42 (1938). 

Lubart, J., in Modern Treatment of Deafness . New York State J. Med., Robert 
Turell, ed. (1963). 

Meniere, M. P. , Gaz. Med. de Paris, 1_6, 55 (1861). 

Miot, C. , Rev. de laryngol. , 1_0_, 49 (1890). 

Morrison, A. W. , Br. Med. J., 2, 345 (1970). 

Nylen, CO., Acta oto- largng. , Suppl. 116 (1954). 

Passow, in Panse, R. , Verhandl. d. deutsch. otol. Gesellsch. , _6, 141 (1897). 

Politzer, A., Ztschr. f. Ohrenh., 25, 309 (1893). 

Rosen, S. , New York J. Med., 53_, 2650 (1953). 



A-8 

Saunders, W. H. , and Paperella, M. M. , Atlas of Ear Surgery , C. V. Mosby Co, , 
2nd Ed., St. Louis (1971). 

Scjeom, J. D. , and Delk, M. T., The Deaf Population of the United States , National 
Association for the Deaf, Silver Spring, Maryland (1 974). 

Schwartze, H. H. , and Eysell, C. G. , Arch. f. Ohrenh. , 7, 157 (1873). 

Shea, J. J., Jr., Memphis, M. J., 33^, 271 (1958), 

Siebenmann, F. , Papers Internat. Otol. Congr. , 9, 207 (1912). 

Sourdille, M., Bull. New York Acad. Med., Jl, 673 (1937). 

Valvassori, G. E. , Amer. J. Roentgen, 94_(3), 566 (1965). 

Wever, E. G. , Lawrence, M. , and Smith, K. R., Arch. Otolaryng. , 48, 19 (1948). 



DESCRIPTION AND ETIOLOGY 
OF OTOSCLEROSIS 



SURGICAL PROCEDURES 



DIAGNOSTIC PROCEDURES 



CONTRIBUTING EVENTS 



1881 von Ttoltsch Gawe otosclerosis Us . 
NR/S/W/P/M 



1893 Polit2er First description ol 
otosclerosis histologv 
NR/S/W/P/M 

1895 Bezold Noted a much greater 

incidence ot otosclerosis 
among females as com- 
pared to males 

NR/S/W/P/M 



1932 von Bekesy Identified mechanism 
of excitation due to 
air and bone conduc- 



- 1876 Kessel Attempted the first stapes 
mobilization and first appli- 
cation of stapedectomv 
NR/S/W/P/M 



- 1888 Boucheron Mobilized 60 stapes 

NR/S/W/P/M 

-1890 Miot Mobilized 200 stapes and 
described the operation in 
detail 

NR/S/W/P/M 

- 1892 Blake Pfacticed stapes extraction 

fof otosclerosis in America 
NR/S/U/P/M 

- 1894 Slacke Stacke's operation in which 

the attic, tympanum, and 
antrum are made into one 

NR/S/ /P/M 

- 1897 Passow Reported the first opera- 

tion for otosclerosis in 
which the ankylosed 
stapes was bypassed 

MR/D/W/P/M 

- 1898 Aldenon Inaugurated technique of 

trephining or fenestrating 
the footplate 

MR/S/U/P/M 

- 1911 Baranv Pioneered in attempts to 

decompress the labvfi'>th 
in otosclerosis. In 1914 he 
received the Nobel Prize for 
his clarification of the 
physiology and pathology 
of the vestibular apparatus 
MR/S/W/P/M 

- 1913 Jenkins Pioneered as did Barany in 

attempts to decompress the 
labyrinth in otosclerosis 
and opened the horizontal 

MR/S/W/P/M 

- 1917 Holmgren Revived interest in the surg- 

ical correction of deafness. 
A pioneer in the fenestra- 
tion operation 

MR/SAW /P/M 

f — 1922 Holmgren Intioduced the use of the 
operating binocular micro- 
scope to otosclerosis 
surgery 

MR/D/W/P/M 



- 1937 Sourdine - Permanently restored- hear- 
ing by means ot fenestra- 
tion tor the first time in 
cases of otosclerosis 

MR/D/W/P/M 



193B Lempert Developed a one-stage 

operation for fenestration 
MR/D/U/P/M 
ACCEPTANCE OF 



-1878 Hartmann Devised the first 
audiometer 

NR/S/ /P/ 

- 1881 Gelle Gelle test described as a 

diagnostic aid for otoscleros 
MR/S/W/P/M 



- 1807 Bozzini Devised an aural speculum 

provided with mirror re- 
flectors and illumination 
NR/S/W/P/M 

- 1841 Holmann Designed the first oto- 

logic head mirror for 
drumhead illumination 
NR/S/ /P/M 

- 1360 von Troltsch Credited with devising 

first modem otoscope 
NR/S/W/P/M 



1908 Bezold Clarified the diffetentiatio 
of conductive and neuro- 
sensory deafness with tun 
fork tests 

NR/S/W/P/M 



-1920 Fletcher Developed an electric 
audiometer which was 
the first commercial 
audiometer 

NR/S/U/P/C 



CODES 

NR Nonmission-Oriented Research 

MR Mission-Oriented Research 

DV Development 

S Significant Event 

D Decisive Event 

U U-S. Event 
W Foreign Event 

G Government Funding 

P Private Funding 

M Medical School - Hospital - 

University 

I Research Institution 

GL Government Laboratory 

C Company 



1943 Guild Otosclerosis histologv "" 

described 

NR/S/U/P/M 
1948 Wever Performed animal enpe'i- ~ 
ments which defined the 
physical properties of 
sound conduction through 
the middle ear 

NR/S/U/P/M 

1961 von Bekesy Was awarded the Nobel - 
Prize lot his discoveries 
concerning the physical 
mechanism ol stimula- 
tion within the cochlea 
NR/S/W/P/M 



FENESTRATION 



- 1953 Bosen Revived stapes 

mobilization 

MR/D/U/P/M 

— 1956 Shea Revised the staped«ctomy 

operation lor otosclerosis 
and added replacement of 
the stapes with a prosthesis 
MR/D/U/P/M 



- 1921 Griessmann Constructed an otoscle- 
rometer tor quantitative 
determination ot the 
Gelle effect 

NR/S/W/P/M 



I 



- 1921 Nylen Was the first to use an 

operating microscope 
(monocular) in ear surgery 
MR/D/W/P/M 

- 1929 Fiemming Initial development of 

antibiotics 

iMR/D/W/P/M 

- 1935 Domack ■ Discovered the antibactefial 

properties of Pronlosil which 
led 10 the sulfa drugs 
NR/S/P/M/ 



'940 Chain Florey purified sufficient 

stable material ol the Penicil- 
lium mold which was later to 
affect significantly the treai- 
ment of infection in Otology 

NR/S/W/P/M 

1941 Abraham Penicillin first used in 
humans 

NR/D/W/P/M 



,1950 Carhart Bone conduction audio- 
metric testing as it 
related to clinical applica- 
tions (Carhart's noichl 
MR/S/U/P/M 



HISTORIOGRAPH - FENESTRATION 



B-1 

APPENDIX B. VENTRICULAR SEPTAL DEFECT 

INTRODUCTION AND SUMMARY 

Ventricular septal defect is the most common of the congenitally occurring cardiac 
anomalies. In the United States, the occurrence of ventricular septal defect is believed 
to range between 2 to 3. 3 per 1, 000 live births. With a birthrate of over 3, 000, 000 per 
year in this country, approximately 6, 000 to 10, 000 babies are born each year with ven- 
tricular septal defect. The mortality due to this defect is estimated at 0.22 per 1, 000 
live births (Hoffman, 1968). This represents approximately 11 percent of those patients 
who are born with the anomaly. Most deaths occur in the first 6 months. 

The term "ventricular septal defect" refers to an opening or communication which 
exists between the left and the right ventricles of the heart. The defect can range in 
severity from a very small hole which presents no serious clinical problem to the com- 
plete absence of a septum dividing the two chambers. Most commonly, the defect occurs 
in the membranous portion of the interventricular septum. Defects in the muscular por- 
tion of the septum are rarer, and frequently are multiple. Muscular defects sometimes 
may be difficult to locate but are not hard to close. A membranous defect may exist 
which brings the left ventricle into direct communication with the right atrium as well as 
with the right ventricle. 

Ventricular septal defect is commonly associated with other congenital cardiac 
anomalies. It is always associated with tetralogy of Fallot and almost always with tri- 
cuspid atresia. Approximately 40 percent of babies born with transposition of the great 
vessels also have a ventricular septal defect. VSD is also occasionally associated with 
coarctation of the aorta, aortic stenosis, and atrial septal defect. 

The clinical manifestations of ventricular septal defect vary considerably, depend- 
ing upon the size of the defect. In cases of a small defect, almost no clinical abnormal- 
ities may be present. On the other hand, a large defect can result in cardiomegaly, low 
weight gain, and frequent respiratory infections, and can lead to congestive heart failure 
and death. An interesting feature of ventricular septal defect with respect to the prog- 
nosis of the patient is that many of the defects close spontaneously before the child reaches 
the age of 6. The spontaneous closure rate is estimated at between 30 and 45 percent. 
It is thus obvious that a distinction must be made between those patients who could develop 
serious consequences because of a ventricular septal defect and those patients who are 
likely to improve with no surgical intervention. 

Present-day methods for management of patients with ventricular septal defect 
represents a triumph of both diagnosis and surgical techniques. As stated before, treat- 
ment of a patient can follow two courses: conservative medical management and surgical 
intervention. The development of accurate and reliable methods for the diagnosis of 
intracardiac anomalies in small infants, plus the accumulation of many data relating the 
prognosis of the patient to the diagnostic findings enable the physician to make intelligent 
decisions regarding the choice of patient management. 

The procedures involved in diagnosis and in surgery for repair of ventricular septal 
defects require a confluence of a great many areas in modern technology. Diagnostic 
procedures are based mainly upon the catheterization of both the left and right sides of the 
heart. In the catheterizations, blood pressures, oxygen saturation, and pH as well as 



B-2 

electrocardiograms and phonocardiograms, may be obtained. Often a radiopaque dye is 
injected for the visualization of the cardiac chambers. The radiographic procedure 
involves much highly sophisticated equipment, including cameras for cineangiography 
and image intensifier s. Most of the surgical procedures for correction of ventricular 
septal defects involve cardiopulmonary bypass in which the blood is taken from the body, 
oxygenated, and then pumped back into the arterial system. An alternative method for 
open-heart surgery involves hypothermia, or cooling the body to a much lower than 
normal temperature to slo'w the normal metabolic processes. The development of both 
of these procedures involves the input of modern technology with respect to the materials 
and monitoring equipment. In addition, it entails an understanding of the basic physio- 
logic processes that are involved in respiration and in cardiac function. During a 
typical surgical procedure, a patient is supported by a heart-lung machine and monitored 
by equipment recording blood pressures, heart rate, oxygen and carbon dioxide levels, 
respiration, and blood acidity. In passing the blood from the body through the device 
and back into the patient, drugs to prevent and control clotting are essential. In addition, 
antibiotics and cardioactive drugs are employed in the procedure. The technology 
involved in blood transfusion, including storage, cross-matching, and compatible mate- 
rials, is an essential aspect of the surgical procedure. In the repair of the defect itself, 
a knowledge of cardiac anatomy and physiology is necessary in order to avoid interfering 
with the conduction pathway in the septum. The postoperative care of the patient also 
involves sensitive monitoring equipment to detect and avoid complications. Because of 
the technological innovations and the experience gained by clinicians, the mortality 
involved in surgical repair of ventricular septal defect is very low. The following his- 
torical account records the important steps by which this significant accoinplishment has 
been achieved. 



HISTORICAL ACCOUNT 



The history of ventricular septal defect is commonly associated with Roger who 
described it in 1879 as being the most frequently occurring of the congenital defects and 
also the most benign. In 1897, Eisenminger described a condition in which a large ven- 
tricular septal defect was associated with high pulmonary vascular resistance and pul- 
monary hypertension. The clinical aspects of the latter condition were certainly not 
"benign" and the condition later became known as Eisenminger' s syndrome. A discussion 
of the history of ventricular septal defect should be viewed within the development of the 
field of clinical cardiology as a whole. In the later 19th century, a great many malforma- 
tions of the heart were described from autopsied specimens. Dr. Maude Abbott, in her 
Atlas of Congenital Cardiac Disease (1936), catalogued all these defects and showed the 
relationship between the anatomical defects and the clinical manifestations. This monu- 
mental work not only presented an analysis of a thousand cases which were seen by her, 
but also brought together a summary of the developments and the comparative anatomy of 
the reptilian, amphibian, and mammalian hearts. As a result of Abbott's work, clinicians 
in the 1930's and 1940's began to diagnose specific congenital heart lesions. Diagnosis of 
cardiac lesions was greatly aided by the development in the 1940' s of the techniques of 
cardiac catheterization, angiocardiography, and indicator dilution curves. 

In the 1940's, the leader in the field of clinical pediatric cardiology was Helen 
Taussig at Johns Hopkins University. Her monograph in 1947 entitled Congenital Mal- 
formations of the Heart stimulated a great deal of interest by physicians around the 
world in the possibility of correctly diagnosing and correcting congenital malformations. 



B-3 

Although Dr. Taussig stated that ventricular septal defects were relatively benign, 
it began to be appreciated in the 1950's that certain types of ventricular septal defects 
were indeed not benign and could be very serious from a clinical standpoint. Cardio- 
megaly, low weight gain, congestive heart failure, and death could result from a large 
septal defect which was uncorrected. With the advent of open-heart surgery in the 
1950's, the question arose as to what types of ventricular septal defects should be cor- 
rected surgically and what types should be managed medically. This question has 
sparked an active controversy for many years. The basis for an ans^wer to this question, 
of course, lies with the establishment of an accurate diagnosis on the nature and extent 
of the ventricular septal defect. As diagnostic procedures improved and more and more 
data were accumulated, it began to be appreciated that many ventricular septal defects 
were indeed benign and could safely be managed conservatively. In fact, studies per- 
formed in 1958 by deCaravalho-Azavedo and Evans, et al. (I960) confirmed the fact that 
many septal defects closed spontaneously. It had been suspected for many years that 
spontaneous closure did occur, but confirmatory evidence was not available until the 
work cited above was published. The estimation of the occurrence of spontaneous clo- 
sure is from 30 to 45 percent (Engle, 1972). Since many ventricular septal defects do 
close spontaneously and many others are benign, a systematic basis xor deciding the 
best form of patient management is essential. The two primary factors which must be 
considered in the classification of ventricular septal defects are the size of the defects 
and the presence or absence of pulmonary hypertension. If the defect is small, rela- 
tively little blood can flow from the left to the right ventricle and thus the hemodynamic 
consequences are minimal. With larger defects, significant left -to-right shunting can 
occur. This causes increased pulmonary ilo'w and a decrease in the efficiency of the 
action of the left ventricle. The pulmonary vascular tree can respond to the continued 
presence of high pulmonary flow by increasing the vascular resistance resulting in a pul- 
monary hypertension. As the pulmonary vascular resistance increases, right ventricular 
hypertrophy can occur and ultimately the right ventricle can override the left ventricle 
and a right-to-left shunt can occur (Eisenminger' s syndrome). It has been observed that 
if a ventricular defect does not close spontaneously, quite often it does not grow in 
absolute size as the child matures. This means that the relative size of the defect with 
respect to the heart becomes smaller as the child grows larger. A systena for classify- 
ing the categories of ventricular septal defects was proposed in 1954 by Selzer. Keith, 
et al. , in 1958 developed a classification system in ■which ventricular septal defects were 
divided into four categories. These classifications helped clarify thinking on VSD and 
served as a basis for a decision as to the form of management. In a recent paper, Engle 
(19V2) reports on a poll of surgeons which states that 95 percent of surgeons do not 
advise surgery when the ratio of the pulmonary flow to the aortic flow is greater than 2:1. 
As will be outlined in succeeding sections, two different approaches to the surgical treat- 
ment of ventricular septal defects are available. The first of these is a palliative pro- 
cedure which consists of banding the pulmonary artery to reduce pulmonary flow. The 
second is a complete surgical correction in which the heart is entered either from the 
right atrium or right ventricle and the defect is repaired. When the defect is small, 
repair can be accomplished simply by suturing the opening together. With larger defects, 
a patch either of a synthetic polymeric material or myocardium is inserted into the 
opening and sutured in place. 

The question of which surgical procedure is indicated is a complicated one. If 
surgical intervention is deemed necessary because of a large defect with significant 
shunting, the pulmonary artery banding procedure has the advantage in a small infant that 
a bypass procedure is not necessary. Placing the band around the pulmonary artery 
restricts blood flow and greatly decreases left -to-right shunting and chances for develop- 
ment of subsequent pulmonary hypertension. However, a second operation is eventually 
needed to correct the defect, at which time the band is removed. The banding procedure 



B-4 

has disadvantages in that as the infant grows in size, the relative amount of pulmonary 
stenosis increases. In addition, the band may also slip from its original position and 
migrate to a position from which it is difficult to remove. On the other hand, a com- 
plete surgical correction may be difficult to accomplish in a very small infant who is in 
poor condition. The palliative artery banding procedure can buy time until the infant 
grows to a larger size and gains more strength. Recently many medical centers around 
the country have reduced the mortality in the total repair to the same level as the mor- 
tality for the pulmonary artery banding (Engle, 1972). Viewed in this context, the direct 
surgical repair is attractive because it avoids the necessity of two separate surgical 
procedures. 

The history of the surgical procedure for the repair of ventricular septal defect is 
closely linked with the entire history of cardiac surgery. In spite of the many advances 
in surgical technique which were made in the first quarter of the 20th century, the heart 
was considered to remain outside the province of the surgeon. The approach to surgical 
repair of defects of the heart was through operations on the vessels leading to the heart. 
A significant step which illustrated that congenital malformations of the heart and sur- 
rounding vessels were repairable came in 1939 (Gross and Hubbard) with the successful 
repair of a patent ductus arteriosis. In this operation, correction of the defect was 
accomplished by simply ligating the vessel between the aorta and the pulmonary artery. 
Prior to the first clinical demonstration, the procedure was developed in dogs and ca- 
davers by Gross in 1939. 

The next nailestone was the work in 1945 by Blalock and Taussig on a method of 
surgical treatment for tetralogy of Fallot. The Blalock-Taussig operation consisted of 
the anastomosis of the subclavian artery to a main branch of the pulmonary artery to 
correct the pulmonary stenosis which is present in the tetralogy of Fallot. The operation 
did not restore the primary defects of the malformation to normal but did allow an 
increased flow of blood in the pulmonary circulation, thus reducing the cyanosis. The 
procedure can best be viewed as palliative and was used in anticipation of procedures 
which subsequently would be developed for a complete correction of the defect. In this 
same year, two groups independently reported on the surgical repair of coarctation of 
the aorta, another congenital cardiac anomaly. Gross and Hufnagel (1945), and Crafoord 
and Nylin (1945), both reported on the successful clinical use of a procedure for removing 
the obstruction to the aortic flow. 

In the following year, Vineberg (1946) reported on a method for surgical treatment 
of coronary artery insufficiency. The operation consisted of attaching the left internal 
mammary artery to the myocardium to provide a blood supply. This technique, while it 
did not constitute repair of a congenital cardiac abnormality, further demonstrated the 
fact that surgical correction of cardiac problems was approachable. In the same year. 
Potts, et al. , improved upon the concept of Blalock and Taussig and developed a method 
for correction of tetralogy of Fallot by anastomosis of the aorta to the pulmonary artery. 

Another step toward operation on the heart itself was made by the development of 
procedures for opening a stenosed pulmonic valve. This procedure was developed in- 
dependently at approximately the same time by Brock (1948) and Sellors (1948) who both 
devised closed techniques for the procedure. A method for the partial repair of ventric- 
ular septal defects was reported by Murray in 1948. Murray attempted closure of the 
defects by inserting a needle through the heart wall and probing for the defect. Sutures 
were placed across the defect and tightened. Murray reported clinical improvement in 
three patients, but had no quantitative data to indicate the degree of closure he effected. 



B-5 

In the following year, a procedure for improving the flow of a stenosed mitral valve was 
described by Bailey (1949). Bailey's procedure was also a closed one in which the 
mitral valve was approached by palpation with a finger of the surgeon. It is interesting 
to note at this point that the first mitral commissurotomy noted in the literature was 
performed 25 years earlier by Souttar (1925). 

The procedure of partially occluding the pulmonary artery to reduce pulmonary 
blood flow in large ventricular septal defects was developed in 1951 by MuUer and 
Dammann (1952). This procedure was undertaken to prevent the occurrence of a left-to- 
right shunt which was often followed by the development of pulmonary hypertension. The 
diameter of the pulmonary artery was reduced to approximately one-third size by a 
polyethylene tape which was wrapped around the artery. 

The first successful repair of an intracardiac anomaly by open surgery was 
accomplished in 1952 by Lewis and Taufic (1953). These clinical scientists repaired an 
atrial septal defect by means of hypothermia and complete occlusion of all vessels lead- 
ing to the heart. This was followed by the first successful repair of intracardiac anom- 
alies using complete heart-lung bypass by Gibbon in 1953. 

The first successful closure of a ventricular septal defect under direct vision was 
performed in 1954 by Lillehei and co-workers (1955). This procedure utilized cross- 
circulation with a human donor for the oxygenation of the blood during the open-heart 
procedure. In the same year, the technique of hypothermia was utilized for the success- 
ful repair of a ventricular septal defect by Bailey. The first successful repair of ven- 
tricular septal defects with the aid of cardiopulmonary bypass was reported in 1956 by 
Kirklin. After Kirklin's report, many medical centers began open-heart surgery, and by 
I960, the procedure was in widespread use. 



Cardiopulmonary Bypass 



One of the essential elements in the surgical treatment of ventricular septal defect 
was the development of a device for cardiopulmonary bypass. Central to this develop- 
ment was a physician. Dr. John H. Gibbon, Jr. , who began work on the device in 1934 
and finally used it on a human for cardiac surgery in 1953, almost 20 years later. For 
the surgical correction of an intracardiac anomaly such as VSD, it is essential that the 
surgical field be clean and free of blood. This necessitates totally bypassing the heart. 
To avoid multiple cannulations and other problems of balancing blood flow, the blood 
bypassing the heart should also be oxygenated before being returned to the body. Thus, a 
device for cardiopulmonary bypass must consist of two elements: a pump (or series of 
pumps) to maintain blood flow and pressure and a system for adding oxygen to and re- 
moving carbon dioxide from the lungs. Dr. Gibbon's concept for such a device was devel- 
oped in 1931 when he was a research fellow at the Massachusetts General Hospital. He 
had the occasion at that time to attend a patient ■who ■was dying from a pulmonary embolus. 
It occurred to him that the patient might be helped if there were some way to remove 
blood, oxygenate it, and then pump it back into the body. He discussed the idea with 
several of his colleagues but received no encouragement. For the next 3 years, he con- 
tinued to think about the idea, but because of other responsibilities of practicing surgery 
in Philadelphia, he was unable to pursue development of the concept. In 1934, he applied 
for a research fellowship at Harvard, and in the fall of that year began work on a heart- 
lung device in the surgical research laboratories of Massachusetts General Hospital. His 
wife also worked with him as a laboratory technician. In terms of present-day research 
programs, the resources available to the Gibbons were quite limited. Much of his 



B-6 

equipment was hand made. The air compressor he used to drive his blood pump was 
obtained in a second-hand store. Dr. Gibbon even stated that when the cats he used in 
the animal studies were in short supply, he captured alley cats in the surrounding 
neighborhood with a gunny sack. 

In beginning his work on the heart-lung bypass device, Dr. Gibbon consulted the 
literature on physiological perfusion to ascertain the best way to oxygenate blood. A 
considerable background existed at that time in perfusion of isolated organs and limbs. 
The concept of an artificial circulation was first described in 1812 by Le Gallois, who 
felt that life could be maintained in any portion of an organism separated from the rest 
of the body if that portion were perfused. The necessity of oxygenating the blood used as 
a perfusate was demonstrated in 1858 by Brown-Sequard. In his experiments, oxygena- 
tion was accomplished by whipping the blood. An improvement on this method was 
devised by von Schroder who bubbled air through a reservoir of venous blood. Further 
improvements on oxygenation were made by Frey and Grube in 1885 who obtained gas 
by spreading the blood as a thin film on the inner wall of a rotating cylinder. In 1895, 
Jacobj oxygenated blood by means of an excised animal lung which was artificially ven- 
tilated. This method of gas exchange greatly reduced the damage to the blood which 
occurred with other methods and much research on heart-lung preparations followed. 

It should be emphasized at this point that these early perfusion experiments en- 
countered several problems, many of which were due to the blood or other perfusion 
media which were used. The early devices contained large reservoirs and often heter- 
ologous blood was used. Furthermore, defibrinated blood was utilized and this caused 
problems with toxicity and internal hemorrhage. At the turn of the century, the incom- 
patibility of blood bet\veen separate species was recognized, and the use of heterologous 
blood was abandoned. 

At the time Gibbon began his work, heparin (see succeeding section) was available 
for use as an anticoagulant and its use eliminated a major roadblock to successful extra- 
corporeal circulation. One of his chief concerns at the time was the selection of a method 
of oxygenation that caused minimal trauma to the blood constituents. He noted that bub- 
bling air through blood caused a serious problem with the formation of foam and so did not 
pursue that approach. The major problem of gas exchange was the attainment of a high 
surface area at the blood-gas interface without causing extensive damage to the blood. 
Gibbon explored several ways to form a thin film and subsequently collect the blood with- 
out foaming or use of high shear rates. He initially consulted ■with a member of the Engi- 
neering Faculty at Harvard, but discarded his suggestion of using the inner surface of a 
revolving truncated cone to form the film because he felt it would be too traumatic. 
Instead, he adopted the use of the inner surface of a vertically revolving cylinder. Blood 
taken from the animal was directed tangentially against the inner surface of the cylinder 
and descended to the bottom under the force of gravity. At the bottom, a stationary cup 
collected the blood which was subsequently pumped back into the animal. In the perfusion 
experiment, a cannula was inserted in the right atrium through the jugular vein. The 
blood was then pumped to the oxygenator, collected, and pumped back into a femoral 
artery. Gibbon simulated a pulmonary embolism by partially occluding the pulmonary 
artery. As proficiency in the procedure was developed and as the equipment was gradually 
improved, the degree of occlusion of the artery was gradually increased. The success of 
the bypass procedure was deinonstrated finally in 1935 when the pulmonary artery was 
completely occluded with the maintenance of normal oxygen tension and blood pressure. 
He was able to maintain the animal for a period of 30 minutes and then reestablish nor- 
mal respiration and circulation for several hours afterward (Gibbon, 1937). Gibbon con- 
tinued to work on the heart-lung device at the Departinent of Surgical Research of the 
University of Pennsylvania. In subsequent work over the next 6 years, he developed 



B-7 

methods for completely sterile bypass so that the effect of the procedure on long-term 
survival of the experimental animals could be determined. In 1939, he reported on his 
studies with a series of 13 cats in which the pulmonary artery was completely occluded 
from 10 to 3 5 minutes. Five of these animals lived for 24 to 48 hours postexperiment; 
four lived from 8 to 23 days; and four survived longer than 1 month. 

Gibbon's work on this device was interrupted by World War II. In 1946, he re- 
sumed his research at Jefferson Medical College. His next objective was to increase 
the capacity of the device so that it could be used on larger experimental animals and 
ultimately with humans. He recognized the need for sound engineering assistance in this 
undertaking since the device was well past the stage of a jerry-rigged apparatus. In this 
regard, he was very fortunate to become associated with Thomas Watson, who was then 
Chairman of the Board of IBM. For the next several years, engineers at IBM assisted 
Gibbon in the design and construction of a device which was large enough to be used with 
humans. The entire cost for the development was borne by IBM. 

In 1950, Stokes and Gibbon reported on their studies with a series of dogs which 
underwent cardiopulmonary bypass. The device still incorporated the vertical revolving 
cylinder for oxygenation of the blood. In the first series of dogs which underwent partial 
pulmonary bypass, all the animals died because of multiple small emboli which formed 
during the perfusion. Therefore, a filter was inserted into the circuit for subsequent 
animals on total bypass. In this series, the animals were on bypass for periods of 30 
to 113 minutes. Eight of the 39 dogs survived, and these were all animals with shorter 
perfusion times. Stokes and Gibbon attributed the deaths to several causes such as acid/ 
base imbalance, anoxia, and hemorrhage (due to improper heparin neutralization). In 
their report, they discussed the potential role of bypass procedures in cardiac surgery 
and indicated that several problems needed solution before clinical use would prove 
feasible. 

In spite of the fact that no published work on total-body perfusion appeared for a 
number of years after Gibbon's 1939 paper, several other workers became active in the 
field in the late 1940' s. In 1947, a group led by Clarence Dennis at the University of 
Minnesota began work on a heart-lung bypass device. They found quite early that the 
rotating cylinder oxygenator designed by Gibbon did not provide adequate oxygenation, 
and they developed another oxygenator ■which consisted of a series of stainless steel discs 
mounted on a horizontal axis. The bottom portion of the discs dipped into a trough of 
blood, and they were rotated slowly, causing a thin film of blood to be distributed on both 
sides of the disc. This method gave much better gas exchange than did the Gibbon device. 
The concept for a rotating disc oxygenator had been developed by Bjork (1948) ■who used 
solid discs attached on a horizontal shaft. 

Dennis and co-workers also experimented ■with various types of pumps in an effort 
to reduce hemolysis and damage to other cellular elements of blood. They also investi- 
gated the use of several different types of drugs to control blood pressure and fight infec- 
tion. A range of oxygen/carbon dioxide mixtures was explored to determine the optimum 
ratio to obtain good gas exchange and at the same time maintain the blood pH at a proper 
level. The addition of sodium bicarbonate to regulate blood pH was also investigated. 
With a view toward performing heart-lung bypass in human surgery, they began experi- 
menting with the closure of artificially created atrial septal defects in dogs, and even- 
tually were successful in obtaining survival. In these experiments, they identified 
several problem areas which needed careful attention in order for the procedure to suc- 
ceed in humans. These included maintenance of adequate blood pressure, maintenance of 
proper arterial O2 tension, removal of venous blood without too much suction, prevention 
of foaming, avoidance of hemolysis, and closure of the heart with the complete exclusion 
of air. 



B-8 

In 1951 after having perfused 80 dogs, the group at Minnesota felt that their pro- 
cedure and techniques had been developed sufficiently to justify clinical application. An 
attempt was made to repair an atrial septal defect in a 6-year-old girl. After the 
patient was placed on bypass and the atriumi opened, it was seen that the defect was 
much more serious than anticipated. A common atriumi was found and an attempt was 
made to close it. During the bypass, another unanticipated problem was encountered in 
that an unusually large amount of blood entered the field through the thebesian vein and 
had to be removed via suction. Approximately 2. 5 liters of blood were replaced, and 
because the blood was citrated, calcium depletion occurred. As a result of these com- 
plications, the patient died. Nevertheless, Dennis and co-workers felt that the bypass 
device had performed adequately and that the procedure had a definite place in cardiac 
surgery. 

In addition to the devices of Gibbon and Dennis, Helmsworth and Clark developed a 
heart-lung device which oxygenated the blood by bubbling oxygen through a column of 
blood. In experimental animals, they addressed the problems of air embolism and 
blood entering the heart from the coronary circulation. In 1952, using total bypass, 
they attempted closure of a ventricular septal defect in a 4-year-old boy. The child did 
not survive, and his death was attributed either to a period of hypotension which had oc- 
curred during surgery or to a cerebral air embolism. 

During this period, Gibbon continued to improve his heart-lung device and replaced 
the rotating cylinder oxygenator by a series of vertical stainless steel screens. This 
innovation had the advantage that it greatly increased gas exchange and had no moving 
parts. Using this device, he experimented with the closure of artificially created atrial 
septal defects in dogs. He was able to decrease the mortality from 80 to 12 percent with 
periods of total bypass of over 30 minutes. 

Gibbon first used the heart-lung machine with a human patient in early 1952. The 
first patient died, not because of any failure of the perfusion procedure, but because 
of a misdiagnosis of the defect. 

In June, 1953, Dr. Gibbon's many years of dedicated effort culminated in his suc- 
cessful closure of an atrial septal defect in an 18-year-old girl. This operation was the 
first successful open-heart procedure in the world with a heart-lung machine (Gibbon, 
1954). 

Of Gibbon's first four clinical cases with complete heart-lung bypass, only one 
patient survived. Nevertheless, he remained optimistic because in each case the device 
had worked well and death was due to extraneous complications. 

The first successful closure of a ventricular septal defect using cardiopulmonary 
bypass was performed by Kirklin and co-workers in 1954 at the Mayo Clinic. In a sub- 
sequent paper (Kirklin, et al. , 1956), he reported on 40 cases, 21 of which involved 
repair of ventricular septal defects. Of these 21 patients, 17 survived, and of the last 14 
cases in the series, 13 survived. Kirklin used a Gibbon-type oxygenator and pumping 
system and was assisted in its construction by Gibbon and personnel from IBM. His 
results were extremely encouraging and demonstrated that cardiopulmonary bypass had a 
definite place in the future of cardiac surgery. 



B-9 
Hypothermia 

The heart-lung machine did not represent the only approach to open surgical repair 
of congenital cardiac anomalies. In the late 1940's, another approach, that of hypother- 
mia, was being developed by Wilfred Bigelow and his group at the University of Toronto, 
The principle behind hypothermia is that lowering the temperature lowers the rates of 
biochemical reactions. Thus, the oxygen requirements as well as the production of toxic 
metabolic products are progressively lowered as the temperature is decreased. In prac- 
tical terms, this means that circulation to tissues can be safely stopped for longer per- 
iods of time at lower temperatures and that the lower the temperature, the longer the 
time period. According to the Arrhenius equation, the relationship between temperature 
and reaction rate is exponential and not linear. Thus, if the rate of a given biochemical 
reaction is reduced by one-half when the temperature is lowered 5 degrees, it will be 
reduced to one-fourth of the original rate when the temperature is lowered 10 degrees. 
As a rule of thumb, oxygen consumption is decreased to one-half at 30 C, to one-third 
at 25 C, to one-fifth at 20 C, and to one-eighth at 15 C. 

Hypothermia is commonly observed in nature in hibernating animals in which the 
body temperature can get as low as 5 C. In nonhibe mating animals, however, tempera- 
tures this low are difficult to attain without special precautions to avoid damage to tissues. 

Bigelow began work on hypothermia for the specific purpose of using this technique 
as a means of achieving open-heart surgery. At the time he began his work, an exten- 
sive background in the effects of hypothermia on animal physiology had been developed 
by numerous workers. He thus had a sound basis for proposing that hypothermia would 
allow the circulation to the heart to be stopped for a period of time during which an open 
intracardiac surgical procedure was performed. As far back as 1875, a report by 
Reinke indicated that humans could survive after the body temperature was lo'wered to 
25 C. The work of Fay in 1940 showed that cancer patients could be cooled to tempera- 
tures as lovi? as 31 C for several days with no apparent ill effects from the procedure. 
The sordid experiments of the Nazis on the ability of humans to withstand cold provided 
some further data on the effects of hypothermia and rewarming on huinan physiology 
(Alexander, 1945). 

The physiological effects of hypothermia in animals had been studied by many 
investigators. It is beyond the scope of the present study to review this work. For 
example. Dill and Forbes reported on the respiratory and metabolic effects of hypo- 
thermia in 1941. The physiology of hibernating mammals was studied by Benedict and 
Lee, who reported that body temperatures as low as 3 C could be tolerated. Cardiac 
changes during hypothermia were followed by Hamilton and co-workers, who noted in 1937 
the lowering of heart rate and reduction in arterial pressure which accompany lower body 
temperatures. Chatfield and co-'workers (1948) studied neurological aspects of reduced 
temperature and noted that nerve action in the rat ceased at 9 C. 

With this background, Bigelow set out to define the conditions of hypothermia which 
could be adapted to open-heart surgery. He used a series of 120 dogs which were cooled 
by peripheral cooling, i.e. , they were placed in a cold room or wrapped in a heat 
exchanger. Anesthesia was given to control shivering until a low enough temperature was 
reached. During the period of hypothermia, several physiological parameters such as the 
ECG, arterial pressure, venous pressure, and oxygen consumption were measured. The 
electrocardiographs were analyzed and the length of various segments as affected by 
temperature were noted. Bigelow found that fibrillation and/or cardiac arrest occurred 
at the temperature range of 16 to 20 C. In several of the animals, he performed a 



B-10 

thoracotomy, clamped off the vessels leading to the heart, and made an incision into the 
heart in order to simulate an open-heart procedure. He experienced some difficulty in 
getting regular contractions reestablished after rewarming and tried electric shock, 
manual cardiac massage, and various drugs. Interestingly enough, the difficulty he had 
in restarting the heart prompted the development by his group of an artificial intra- 
cardiac pacemaker which was years ahead of its time. 

The results of Bigelow's studies were sufficiently encouraging to stimulate several 
other investigators. In 1951, Boerema published results of his studies with intracardiac 
procedures in dogs with the aid of hypothermia. He was able to interrupt circulation 
safely from 10 to 20 minutes, during which time the heart was entered. In the same 
year, Lewis and Taufic at the University of Minnesota began working with hypothermia 
in a series of dogs. They created artificial atrial septal defects in dogs under condi- 
tions of hypothermia. Twenty-seven of 39 dogs survived the procedure. Twenty-six of 
the survivors were reoperated upon for closure of the defect, and, of these, 17 success- 
ful closures with survival were obtained. 

These results were so successful that Lewis and Taufic felt a clinical application 
was warranted. On September 2, 1952, an atrial septal defect in a 5-year-old girl was 
successfully closed using the technique of hypothermia. The most serious problems 
identified were ventricular fibrillation and coronary air embolus. 

The technique of hypothermia was enthusiastically adopted by Swan who carefully 
evaluated the causes of operative failure. In a series of dogs, he developed a procedure 
in which they were cooled with ice water to 20 to 25 C. Blood flow was stopped for 
periods as long as 25 minutes. To prevent and/or counter fibrillation, Swan hyper- 
ventilated to reduce carbon dioxide levels and administered potassium chloride if 
necessary. Air embolism of the coronary arteries was avoided by filling the chest 
cavity with Ringers solution before closing the heart. Swan reported in 1953 on a series 
of 15 human patients on whom a variety of cardiac anomalies were repaired under direct 
vision during hypothermia. Of these, only one death occurred. He felt that the success 
of this procedure warranted a larger clinical study. 

With the exception of Boerema, all of the above investigators used the method of 
peripheral or whole-body cooling to lower body temperature. This nnethod has several 
disadvantages. For example, the shivering which is induced by the cold must be coun- 
tered by an anesthetic. Secondly, since the cooling is from the outside inward, consider- 
able time is required before the internal organs, such as the heart, brain, liver, and 
kidneys, are cooled to the desired temperature because of the body's insulating system. 
Similarly, long periods of time are required for rewarming after the procedure. An 
alternative to peripheral cooling which offers several advantages is the technique of 
"core" cooling. In this method, the patient is cooled by passing the blood through a heat 
exchanger in the extracorporeal circuit. This method is much more rapid and efficient 
than peripheral cooling and the shorter time is of benefit to the patient. As the cooled 
blood is returned to the patient, the organs with the highest perfusion rate (heart, kidney, 
brain, and liver) are cooled the fastest. Since these organs normally have a very high 
oxygen uptake, compared with the rest of the body, the oxygen demand can be reduced 
very rapidly. The method also allows for better control of body temperature. As soon 
as refrigeration of the blood is stopped, the heat stored in the skin, muscles, and bone 
causes rewarming. The technique of blood stream cooling to induce hypothermia was 
described in 1921 by Heymans. Its adaptation for hypothermia in conjunction with cardiac 
surgery was utilized in 1951 by Boerema, and further developments were made by several 
other investigators including Peirce and PoUey (1953) and Senning (1954). The first 
clinical use of core cooling was reported in 1955 by Brock and Ross. 



B-11 

Since the inception of hypothermia in the early 1950' s, a great many variations of 
the technique have appeared and are too numerous to describe here. Core cooling has 
been combined v/ith peripheral cooling, for example. Various modes of cooling, such as 
arteriovenous pumping, arterial-arterial pumping, venoarterial pumping, and veno- 
venous pumping, were used. For deep hypothermia, it is necessary to take over me- 
chanically the pumping of both ventricles. The technique of autogenous lung oxygenation 
for deep hypothermia was developed by Drew, et al. , and involves cannulating both sides 
of the heart and allowing the lungs to oxygenate the blood during the cooling-down period. 

Although the two procedures of heart-lung bypass and hypothermia came into 
clinical practice at approximately the same time and both allo'wed repair of intracardiac 
anomalies, they were at first felt to be mutually exclusive. Lillehei in 1955 felt that 
hypothermia could cause irreversible damage to the A-V conduction pathways. Over the 
next few years, however, it appeared that there were definite advantages in combining 
the two techniques. Because the metabolic rate of the tissues is lower, the oxygen 
demands are not as great. At the present time, the use of moderate (30 to 32 C) hypo- 
thermia during cardiopulmonary bypass is in widespread clinical practice. 

With the success of open-heart surgery which was reported in the early 1950's, 
many medical centers around the world recognized the need to develop the capability for 
cardiopulmonary bypass. This presented problems in obtaining the necessary equipment 
and in training the personnel in operating the equipment. The pumps (roller or peristal- 
tic) were available from several sources commercially. However, the complete device 
including the oxygenator, reservoir, and safety controls was not available. Generally, 
such devices were made in the machine shops associated with large medical centers. 
During the latter portion of the 1950's, many improvements were made in oxygenator 
design relating to the ease with which the device could be dissembled, cleaned, and 
sterilized. A simplification which eliminated the need for the cleaning and sterilizing 
procedure was made by Gott in 1957, who developed a disposable plastic sheet oxygenator. 
This device, which was a bubble-type oxygenator, consisted of a vertical oxygenating 
column, a debubbling chamber, and a stainless steel mesh filter. It served as a proto- 
type for the disposable oxygenators which are marketed by several companies and are in 
wide clinical use today. 

The materials of ■which extracorporeal devices are constructed are of considerable 
importance in determining the amount of damage inflicted upon the blood. Contact of 
blood with a foreign surface can result in activation of the coagulation sequence, activa- 
tion and removal of platelets, denaturation of plasma proteins, and sublethal damage to 
red blood cells. The extent of damage is dependent in part on the surface properties of 
the materials with which blood is contacted. Early physiologists investigating perfusion 
used many materials which were traumatic to blood components and the recognition of the 
importance of selecting proper materials made possible the advances in perfusion. 
Several of the synthetic polymeric materials developed in the 40's and 50's find consider- 
able use in extracorporeal devices. Silicone rubber available in a medical grade (see 
case study on the cardiac pacemaker) has a relatively low level of deleterious interactions 
and low level of leachable material. Silicone rubber has properties which make it 
desirable for use in peristaltic and roller pumps. Plasticized poly(vinyl chloride) also 
finds extensive use in extracorporeal circuits because of its flexibility and transparency. 
Teflon® is used for cannulae, connectors, and many other components. In addition, poly- 
ethylene, polypropylene, polycarbonate, and nylon also find extensive use. 

When bubble oxygenators are used, the foaming of blood can produce serious prob- 
lems unless an antifoaming substance is used. Silicone antifoaming agents serve very 
well for this purpose since they are highly effective and are compatible with blood. These 



B-IZ 

substances placed on the surfaces of the components of the bubble trap in oxygenators 
eliminate the problem of excessive foaming. Other silicon derivatives which are used 
are the silanizing agents which form a low surface energy (nonwettable) coating on 
materials such as glass and metals, thus making these materials more compatible with 
blood. 

Many devices, such as cannulae, suction tips, and screen oxygenators, necessitate 
a metal contacting blood. The material of choice is stainless steel because of its ability 
to be highly polished and its low chemical reactivity. 

From the above, it can be seen that a necessary link in the development of the 
heart-lung machine was dependent on the materials which became available in the mid- 
20th century. 



Cross -Circulation 



Another method for achieving open-heart surgery which appeared briefly in 1954- 
56 was the technique of cross -circulation. Dr. C. W. Lillehei and associates at the 
University of Minnesota used the lungs of another living human to serve as biological 
oxygenators for the patient's blood. In the circuit, the superior and inferior vena cavae 
of the patient were cannulated, passed through a peristaltic pump, and into the saphenous 
vein of the donor. Blood was removed from the donor via the femoral artery, passed 
through a pump, and then back into the patient's aorta. Dr. Lillehei used relatives of 
the patients as donors and the donors were selected on the basis of homology with respect 
to the ABO system. An attempt was also made to obtain matching with respect to blood 
cell types. Lillehei utilized a principle developed by Andreasen and Watson (1952) called 
the "azygous principle" which held that the flow required for perfusion could be less than 
the normal cardiac output. In a series of publications in 1955, Lillehei and co-workers 
reported on the surgical correction of ventricular- septal defects, tetralogy of Fallot, 
pulmonary atresia, and atrioventricularis communis in a total of 50 patients. The results 
were highly successful. Nevertheless, in the next year, he reported on the use of a dis- 
posable oxygenator rather than a human donor in the extracorporeal bypass circuit. The 
increased reliability and safety of artificial oxygenators had been sufficiently demon- 
strated at this time so that the risk associated with the use of a human donor for oxygena- 
tion could no longer be justified. 

Another approach to cross-circulation was taken by W. T. Mustard, et al. , in 
1954 who used the lungs of a monkey to serve as the biologic oxygenators. Mustard 
operated on a group of seven infants, ranging in age from 19 days to 2 years, for the 
surgical correction of the transposition of the great vessels. The length of time of bypass 
through the monkeys' lungs varied from 10 minutes to 3-3/4 hours with the average time 
being around 2 hours. None of the babies survived. The monkey lungs performed well 
with respect to maintaining oxygen tension and did not appear to damage components of 
the blood. Mustard felt the procedure could be successful in infants with less serious 
malformations. However, the method was not studied further, probably because of the 
advent of successful artificial heart-lung procedures. 



B-13 
Anticoagulation 

One of the technological developments which was absolutely essential for the suc- 
cess of open-heart surgery was an effective, safe anticoagulant for the blood. When 
blood comes in contact with a foreign surface, stimulation for coagulation results. Thus, 
in an extracorporeal device which pumps and oxygenates the blood, the abundant surface 
area of the device could provide an adequate stimulus for coagulation which would result 
in clot formation and would clog the device. Worse yet, blood may be activated by the 
device flow back into the patient, and subsequently thrombose in the patient's vascular 
system. For effective operation, an anticoagulant is necessary which prevents clotting 
in the extracorporeal circuit, and which subsequently can be neutralized as the blood is 
returned to the patient so that hemorrhage is avoided. In addition, the anticoagulant 
should be safe, reliable, and not have untoward aftereffects. Heparin, the anticoagulant 
drug which was used by Gibbon and is still used today, meets these requirements, and 
its discovery and commercial availability provided a positive stimulus in the develop- 
ment of open-heart surgical techniques. 

Heparin was discovered accidentally in 1916 by Jay McLean who was a second-year 
medical student working in the laboratory of William Howell at Johns Hopkins University. 
He was given the task of isolating and purifying components of thromboplastic liver 
extracts in hopes of finding a "thrombokinase" which was a primary activator of coagula- 
tion. One of the fractions he isolated had a pronounced coagulation-inhibiting effect 
instead of a procoagulant activity. Howell was quick to recognize the potential thera- 
peutic value of this substance, and the next year described it in his Harvey Lecture. He 
named the substance "heparin" because it ^vas originally extracted from liver. Howell 
continued to work with heparin and found that it was present in many organs other than 
the liver. He developed improved methods for heparin isolation, and as more nearly 
pure samples were available, he discarded his original idea that heparin was a 
phosphatide. 

In 1928, he published a detailed account of his work on the isolation and properties 
of heparin, and this stimulated interest in several other laboratories around the world. 

A crude preparation of heparin for experimental purposes was available commer- 
cially as early as 1922 from the Hynson, Westcott and Dunning Co. of Baltimore. 

About this same time, studies on the in vivo effects of heparin were iinder way in 
several laboratories. Reed in 1928 gave large amounts to dogs to show its efficacy and 
safety. It was found that the undesirable side effects of heparin preparations came from 
the impurities. Thus, an efficient method for preparation of a relatively pure heparin 
■was essential before the drug could come into widespread clinical use. Such a method 
was developed by Charles and Scott, working at the Connaught Laboratories of the Uni- 
versity of Toronto. In 1933, they described a preparation method from beef liver or lung 
which gave a high yield of heparin in a purer form than was previously available. Their 
method was subsequently improved considerably in 1936 by the use of a precipitation 
employing a benzidine salt. The heparin preparation derived by this method gave very 
little ash and in the form of a barium salt was pure enough to be used as a standard for 
heparin. Charles and Scott's method serves as a basis for commercial preparation by 
many pharmaceutical houses around the world and several good heparin preparations 
became commercially available in the late 1930' s. 



B-14 

Demonstration of the clinical safety and efficacy of heparin was first done by 
Hedenius and Wilander in 1936. These investigators gave repeated doses of heparin 
intravenously to study its effectiveness in controlling postoperative thrombosis. They 
found that the more nearly pure the heparin, the fewer the side effects, and that effica- 
cious doses of heparin could be administered without excessive danger to the patient. 
A great stimulus to the clinical utilization of heparin was provided by the publication of 
a monograph on heparin in 1939 by Jorpes. This book served to acquaint the medical 
community with heparin by providing a comprehensive background on its composition, 
mode of administration, and clinical effectiveness. 

In 1938, work by Chargaff and Olson provided the final necessary key to the suit- 
ability of heparin for use in extracorporeal circulation. These workers demonstrated 
that the anticoagulant activity of heparin could be neutralized by protamine. The use of 
protamine clinically to counteract hemorrhage which could result from administration of 
large amounts of heparin followed soon after (Jorpes, 1946). In the case of extracorpo- 
real circulation, heparin could be added to the blood as it exited from the body and then 
could be subsequently neutralized with protamine sulfate before reentry. 

Diagnosis 



An essential element in the treatment of congenital cardiac abnormalities is an 
accurate diagnosis of the nature and the seriousness of the defect. With respect to VSD, 
an accurate diagnosis is particularly important because the size of the defect forms the 
basis for deciding whether to perform surgery or to manage the patient conservatively. 
Several procedures are utilized presently to diagnose cardiac anomalies, and a detailed 
description of each of these methods is beyond the scope of this report. These methods 
include analysis of heart sounds, analysis of electrocardiograms. X-ray, and a host of 
techniques which are based on catheterization of the heart. The existence of a cardiac 
murmur has long been associated with a VSD and is probably the first symptom which 
alerts the physician to perform further diagnosis. The murmur is, of course, related 
to the size of the defect and to the absence or presence of pulmonary hypertension. 
Analysis of heart sounds by a phonocardiogram can aid in determining the nature of the 
defect. 

A description of abnormalities associated with VSD as shown by radiography v/as 
first presented in 1913 by Vaquez and Bordet. With a moderate to large VSD, the X-ray 
shows peripheral and central vessel enlargement of the pulmonary vascular tree. If a 
substantial left-to-right shunt exists, left ventricular hypertrophy may be present. 

Electrocardiographic indications are affected by the size of the defect, the presence 
or absence of pulmonary hypertension, the degree of left ventricular overload, and the 
pressure overload of the right ventricle. A discussion of the complexities of the electro- 
cardiographic analyses is beyond the scope of this review. A few generalizations can be 
made, however. Often the QRS electrical axis is directed downward. The P-waves are 
indicative of left ventricular overload. 

The most fruitful diagnostic tool for detection of cardiac anomalies is the catheter- 
ization of the heart. When a catheter is introduced into the heart, measurements of 
pressure, flow, oxygen tension, dilution of indicator substances (dyes, gases, etc.), 
and radiopacity of injected dyes can be made. The technique of intracardiac catheter- 
ization, which ultimately revolutionized diagnosis and evaluation of cardiac function and 
disease, was introduced by Forssmann in 1929. Forssmann's objective in exploring 



B-15 

methods to catheterize the heart was not to devise a diagnostic tool but to find a way to 
administer safely an intracardiac injection to stimulate the heart during cardiac arrest. 
Intracardiac injection was highly controversial at that time. Although it was effective 
in some cases, considerable risk was involved because of the danger of cardiac tam- 
ponade, danaage to the coronary vessels, and pneumothorax resulting from the pene- 
tration of the chest wall, pericardium, and heart wall. Forssmann sought to circum- 
vent the danger by inserting a catheter to administer the drug. Ironically, as was pointed 
out a few years later by Hyman (1930), it was the physically traumatizing action of the 
needle itself and not the pharmacological action of the drug which stiraulated the heart 
and reversed cardiac arrest. * 

After analysis of the appropriate anatomical relationships, Forssmann decided 
the best route would be through the peripheral venous system. Accordingly, he inserted 
a urinary catheter into a vein in the right arm of a cadaver and advanced it until he en- 
countered resistance. He was able to show that the tip of the catheter was in the right 
ventricle by dissection. He next inserted a catheter into a right arm vein of a colleague, 
but did not advance it all the way to the heart because of the apprehension of the patient. 
Because of a lack of further volunteers, he then inserted the catheter in a vein in his 
own left arm to a length of 6 5 cm. To determine the location of the catheter, he walked 
to the radiography department and had himself X-rayed. Forssmann reported on the use 
of intracardiac catheterization to administer drugs in one patient who was in severe circu- 
latory distress. Upon the infusion of epinephrine, the patient's pulse became stronger, 
but he died a few hours later. It is ironic that the tremendous therapeutic benefit which 
did result from Forssmann' s work was not because of intracardiac drug injections, since 
they were abandoned a few years later. Forssmann followed up his first report by a sec- 
ond paper in 1931 in which he described the visualization of the right chamber of the 
heart by means of an X-ray contrast substance. 

The potential value of cardiac catheterization for diagnostic purposes was recog- 
nized by Cournand and Ranges, who developed a procedure in which the cannula was con- 
tinuously purged with a saline solution to prevent thromboses at the tip or in the lumen 
of the catheter. They reported in 1941 on eight experiments in humans in which they 
withdrew blood from the right atrium for gas analysis. The procedure was safe and the 
patients experienced no pain. 

Specific applications of cardiac catheterization to diagnosis of cardiac anomalies 
were developed by Dexter, et al. , in 1947. In a series of papers, these authors de- 
scribed a method for catheterization of the right atrium, right ventricle, and pulmonary 
artery. The pressure and oxygen saturation of the blood in these locations were mea- 
sured in a series of control patients. Then, Dexter measured the same parameters in a 
series of patients -with known cardiac anomalies. He made some observations v/hich form 
the basis for diagnostic procedures as they are practiced today. For example, he found 
that in patent ductus arteriosis, blood in the pulmonary artery is more highly oxygenated 
than in the right ventricle. In VSD, the blood in the right ventricle has a higher oxygen 
content than that in the right auricle. ASD may be detected by introducing the catheter 
through the defect into the left atrium. The pulnaonary stenosis associated with the 
tetralogy of Fallot can be sensed by the higher pressure found in the right ventricle com- 
pared with that in the pulmonary artery. 

In present-day diagnostic procedures, right heart catheterization remains the 
basic starting point. The pressure and oxygen saturation measurements outlined by 
Dexter are still made; however, the apparatus which is involved has undergone 



'Hyman used this deduction as the basis of developing an artificial cardiac pacemaker. 



B-16 

considerable refinement and improvement. Catheters which are specially designed to be 
easily steerable are available. Miniaturized pressure transducers which fit on the cath- 
eter tip are often used. Electrodes for obtaining intracardiac electrocardiograms and 
miniaturized microphones for intracardiac phonocardiograms are available. Oxygen 
levels of blood can be measured by an electrode or by a photometric technique, and 
these methods allow multiple readings of oxygen saturation to be made conveniently. 

In addition to pressure measurements, indicator dilution measurements are often 
made. In 1953, Swann and Wood introduced the use of Evans blue as a dye and this proce- 
dure provided a ready means for dye measurement in the blood. In the simplest applica- 
tion of this technique, a dye is injected into the right atrium. Blood is then withdrawn 
from a peripheral artery into a flo'w photometer which measures the level of the dye in 
the blood. The shape of the curve of dye concentration plotted against time is diagnostic 
of intracardiac shunts. Sensitive detection of even small left-to-right shunts can be 
made by hydrogen gas technique developed by Clark and Bargeron. In this technique, a 
small amount of hydrogen gas is inhaled through a mask by the patient. Hydrogen con- 
centration in the right atrium or right ventricle is measured by a platinum electrode 
which is introduced via a catheter. The premature presence of hydrogen in the right 
chambers indicates a left-to-right shunt and the concentration of hydrogen is indicative 
of the size of the defect. 

In addition to the right heart, catheterization of the left heart also serves as a 
diagnostic procedure. When a septal defect exists, the left side of the heart may be en- 
tered through the defect. The left heart may also be entered via retrograde catheteriza- 
tion of a peripheral artery. This method of catheterization was first proposed in 1950 
by Zimmerman. The present widespread acceptance of the approach from the femoral 
or right brachial artery is due to the studies at NIH by Morrow, Braunwald, and Ross 
reported in I960. 

The sophistication and diversity of the equipment used in present-day diagnosis of 
cardiac anomalies give ample indication of the application of modern technology in med- 
ical practice. It would be impossible to describe the methods or equipment presently in 
use within the confines of this report. Instead, we have attenapted to outline the princi- 
ples and simple beginnings upon which the present technology is based. 



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B-17 
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B-18 

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Goor, D. A. , et al. , Chest, 58_, 468 (1970). 

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(1937). 

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B-19 

Heymans, J. F. , Arch. Inst. Pharmacodynam. , 25, 1 (1921). 

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B-20 

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ANTICO 

1916 McLean I 
I 



1917 Howell C 
P 

H 



1922 Howell C 



1928 Howell 



1933 a 

a 



1935 



/ 
193' 



ANTICOAGULANTS 



MR/S/U/P/M 
1922 HovwII Developed lim mWhod lor 

MH/S/U/P/M 

1928 Howoll Improved meihotti ot heparir 
iMlalion, Lloyds 'eageni 
omploved 

MR/S/U/P/M 



1933 Diiiles Oevsloped tiril method lot 

Scoti lergo-icalu pteparalion at high 
purily heparin 

MR/0/W/G/M 

1935 Jarpcs KBrolinska Inniiuie, Stockholm 
Analviit ol heparin structure 
NR/S/W/G/M 



MR/S/W/G/M 
1936 Hedoniui Demonstrated ellicacy and 
safely of heparm in humans 
MR/D/W/G/M 



CARDIAC CATHETERIZATION 



1929 Fodsmann Caihaterlied heart by ii 

MR/0/W/P/M 

1931 Fortsmann Infected conttait mediu 

chamber 

MR/D/W/P/M 



1938 Robb Developed practical method 
Steinbeig of angiography 

MR/S/U/ / 



1941 Coucnand Developed delir>iiiva tech- 
flanges niquei of right heart 

catheterization for diagnosit 
MH/S/U/P/M 



1947 DoHtei Developed system for charai 



Technique ol li 



1953 Swan Use of Evani blue in dye 
Wooddilulion curves 

MR/S/ / / 



COOES 

NR Nonmiislon-Oriented Research 

MR Mission -Oriented Research 

DV Development 

S Significant Event 

Decisive Event 
U US, Event 

W Fo'eign Event 

G Government Funding 

P Private FuntJing 

M Medical School - Hospital — 
Univeriity 

1 Reseafch Institution 
GL Government Laboratory 
C Company 



I ClBik Hydro^n gas moth' 



MR/S/U /G/M 

1960 Mariovv Procedure lor loll heart 



HEART LUNG BYPASS 

Extensive background in perfusion 
organs, limbs, anid whole body 
developed in 19th century 



SURGICAL PROCEDURES 



HYPOTHERMIA 



1921 Huyrtwns Induced hypolheimia 
anintali by blood ilrci 

NR/S/ / / 



. 1934 GibtKin Begins work on catdiopul- 
monetv bypass device 
MR/D/U/P/M 



1936 Gibbon Succesilully suppotli life in 
animal with comploie cardio- 
pulmonary bypass 

MR/D/U/P/M 



1940 Gibtion Studied neurological effect 
Weint>erger ol cardiopulmonary bypass 
MR/S/U/P/M 



} Gibtion Gibbon begini inlofmal — 

MR/D/U/P/M 

i ejork Develops rotating disc OKygenalof — 

MR/D/ / / 
9 Dennis Heart lung bypass device with — 

1 rotating disc screen. First 
human patroni 

MR/D/U/P&G/M 
Q Gibbon Developed procedures for — 

Stokes intiBcardiac repairs during 

MR/S/U/P/M 

3 Slokos Oevelopmont o( cylinder screen — 
Flick oxygenator 

MR/S/ / / 
D Sitak Development of left heart pump — 

MR/S/U/G/M 

2 Millei Develop iiationary screen _ 
Gibbon oxygenator 

MR/S/U/P/M 

; Helmiwo'th Developed bubble — 

Clark OJiygenatoi 

MR/S/U/G/M 



1957 Gott Disposable plonic bubble 
oxygenator 

MR/D/U/P&G/M 



MR/S/U/G/GL 



1939 Gross Repair of patent ductus 
Hubbard aiietioiis 

MR/D/U/P&G/M 



1945 Giost Suraical repair of coarctatior 
Hulnagel of the aorta 
Ctaloord 
Nylin MR/D/U/P&G/M 



1946 Potts Corn 



Surgical method lof treatment 

MR/D/U/P&G/M 

n of Fallot's tetralogy 



1946 Vineberg Anaitomijit ol internal mam- 
mary anery lo myocardium 
lor repair of coronary iniuf- 

MR/D/W/G/M 



1946 Murray Blind 



MR/D/W/G/M 



MR/D/U/P&G/M 



1949 Bailey Widening ol mitral valve 
MR/D/ / /M 



1S50 elum Developed procedure of 
Migebow cross-ciiculatlon in animal 

MR/D/U/G/M 



restriction ol pulrnonar^ 

MR/D/U/G/M 



1940 Fay Cooled cancer patients to 31 C 

MR/S/U/P/M 

1941 Dell Respltaiory and motstKilic 
Fotbes ellects ol hypothetmia 

NR/S/ /M 



1953 Gibbon Perli 



hum; 



US lirst successlul ope 
urgicsl procedure on 



MR/D/U/P/M 

1954 Mustard Attempted repair ol Iran; 






MR/S/W/G/M 



1954 Bailey Ventricular septol delect closed - 
succesilully with hypothermia 
MR/S/U/G/M 

1956 Kirkiln First successful closure Ot VSD ' 
using heal lung bypatt 

MR/D/U/P&G/M 

19S8 Trues Woiked out conduchon path- 

Bishol nays in system and provided 

knOMledga m avoiding igtro- 



1943 Chatlield Studied nerve action in 
hypothermia 

NR/S/U/ /M 



1950 Bigeloiv Developed methods loi In 
MR/D/W/G/M 



1951 Bocrema Developed intracardiac pro- ■ 

hypothermia 

MR/S/W/G/M 
1952 BIgelotv Developed lunhor mtroco'diac- 

MR/S/W/G/M 

1952 Lewis First human success ivith hypo- - 
Taulig thermia closure Ol ASO 

MR/D/U/P&G/M 



MR/S/U/P&G/M 

1953 Pei'co Advanced use ol core cooling 
Policy in hypothermia 

MR/S/U/G/M 



1959 Drew Method of deep hypolheimU b 
cannulsiion of t>oth iitJes ol 
hwn 

MR/S/W/G/M 



HISTORIOGRAPH - VENTRICULAR SEPTAL DEFECT 



C-1 
APPENDIX C. KIDNEY TRANSPLANTATION 

INTRODUCTION AND SUMMARY 



By the end of 1975, there wiLL have been close to 20, 000 kidney transplants per- 
formed. This is based upon the fact that a recent Transplant Registry reported I, 660, 
2, 164, and 2, 207 kidney transplants in the United States for 1971, 1972, and 1973, 
respectively, and at the end of 1972, over 10, 555 renal transplants had been reported 
to the Registry. At the same time, it is estimated that close to 10, 000 patients per 
year become candidates for treatment by maintenance hemodialysis or renal trans- 
plantation (Kountz). 

As recently as 1972, the percentage of living donors (as opposed to cadaver 
donors) was approximately 40 percent of the total donor population and it is hoped that 
the living donor can eventually be replaced completely. This, of course, will depend 
upon improvements in cadaver organ procurement, refinements in storage, and devel- 
opments in the control of the immunologic process in the recipient directed against 
the graft (Bergan). 

With the present methods of general immunosuppression it is estimated that 
approximately 70 percent of recipients of kidneys from close blood relatives have 
functioning grafts at 2 years while for recipients of unrelated cadaver kidneys the 
figure is 50 to 60 percent (Calne, 1974). This is an unusual statistic in that histocom- 
patible donor- recipient matches should provide a much better (statistically) chance of 
organ acceptance (as compared to nonhistocompatible matches). Terasaki et al. (1974) 
have determined, using several European as well as United States transplant registries, 
that the incidence of cadaver graft survival at 1 year is approximately 50 percent for 
the best possible four antigen matches and also approximately 50 percent for the worst 
possible antigen matches. This indicates that with the present level of general immuno- 
suppressive therapy there will be no significant increase in these figures without the 
advent of some applicable method of donor-specific immunosuppression. 

The initial impetus of renal transplantation occurred in the early 1900 's when 
Carrel developed the techniques for vascular suturing. In these early attempts, renal 
transplantation was tried in both humans and animals. The early attempts in humans 
involved heterografts only and there was essentially no success obtained from these 
trials. The dog homograft experiments were equally disappointing although Zaaijer 
in 1908 performed a successful long-term autotransplant (4 years). At this time the 
investigators were beginning to realize that an autograft had a better success rate than 
did a homograft. All these transplant attempts tended to draw to a close when three 
investigators (Williamson, Ibuka, and Holloway) confirmed by microscope the changes 
which were taking place in the kidney, i.e., plasma cell infiltration, tubular necrosis, 
and lymphocyte infiltration. 

After 1925 there were a few transplants being attempted in dogs and a few unsuc- 
cessful attempts at renal homotransplantation in man, using cadaver donors, between 
1936 and 1951. However, the first transplants to achieve sufficiently good function, 
in one case up to 6 months, was the clinical series using cadaver kidneys performed by 
Hume and his associates in 1952-1955. Following this initial success a mother-to-son 
transplant was reported by Michon in 1953. The significance of this transplant was 
that it functioned immediately without the normal acute tubular necrosis seen in cadaver 



C-2 

transplants. However, this kidney transplant eventuaLLy was rejected 22 days Later 
(Beathard). 

The first identical twin transplant was then reported in 1956 by Merrill and his 
associates in which the patient survived for over 8 years before dying of a myocardial 
infarction. A significant development occurred during these transplants which were 
conducted in Boston at the Peter Bent Brigham Hospital. That was the advent of the 
artificial kidney for treating chronic uremia via dialysis. In addition, the artificial 
kidney could now be used in transplant cases if the transplant suffered from initial 
failure, i.e., anuria. 

Finally, in 1959 and I960, the first reports of nonidentical twins being transplanted 
were made by Hamburger in France and Merrill in the United States. It was also 
during this period that methods of immunosuppression were seriously considered. The 
application of these methods grew from two sources. First was the basic work of 
Gibson and Medawar (1943) and Medawar (1944-1948) describing the immunologic pro- 
cess of skin graft rejection and second was the clinical realization of and need for 
immunosuppressive therapy. 

Extensive animal experimentation on the application of immunosuppression was 
begun in the early 1950's, soon after completion of Medawar's findings. Using this 
work as a basis. Hamburger in France in 1959 significantly prolonged a human renal 
homograft by using whole body sublethal irradiation as a method of immunosuppression. 
Radiation immunosuppression was soon abandoned for general use because of reports 
of severe bone marrow depression and frequency of systemic bacterial sepsis. At 
this same time (1958-1960) individuals at Burroughs Wellcome and Company, Inc. in 
England had synthesized and were testing the biological activity of 6-mercaptopurine 
(6-MP). In 1959, Schwartz and Dameshek found that 6-MP induced immunologic toler- 
ance in rabbits. There followed a rapid series of experiments and publications in 
which 6-MP was tested as an immunosuppressant for renal grafts in dogs (Calne, I960; 
Zukoski et al. , I960) and then in human renal homografting (Kuss et al. , 1961) with 
some success. Coincident with this work was the synthesis of azothioprine (a 6-MP 
derivative) by Elion et al. at Burroughs Wellcome in 1961. Calne conducted successful 
canine trials of azothioprine in the same year and Murray et al. , successfully applied 
azothioprine in human renal transplantation the following year'(1962). 

Based on the animal work of Germuth and Ottinger (1950), Billingham et al. 
(1957), and Baker et al. (1952), Goodwin et al. determined in 1962 that steroid com- 
pounds were extremely useful in reversing rejection crisis in human renal transplant 
recipients. Starzl et al. applied both azothioprine and prednisone (a steroid) in a 
logical clinical regimen using azothioprine as a constant immunosuppressant and pred- 
nisone to reverse rejection crisis in 1963, thereby establishing the basis for the clini- 
cal regimens used today. In 1967 Starzl et al. used antilymphocyte serum (ALS) for 
the first time in a clinical setting and thus added the last statistically successful method 
of immunosuppression for current application to renal transplantation. 

The other major areas of transplantation which have as yet not yielded full clinical 
potential are the areas of immunogenetics and antigen recognition. It is important to 
briefly include the milestones in these areas in the historical account because they 
provide 

(1) Definition of the histocompatiblity antigens 



C-3 

(2) Tests useful in donor matching 

(3) The potential approach for specific immunosuppression and 
eventual 100 percent success in renal transplantation. 

A few heterotransplants have since been performed as a result of immunosuppressive 
therapy, but the longest functioning transplant (chimpanzee) has been 2Z weeks. It 
would appear that heterotransplantation must await a more definite approach of immuno- 
suppressive therapy. In addition, the question of a sufficient supply of chimpanzees 
and baboons must be resolved before heterotransplantation will have a future in human 
kidney transplantation. 

Renal transplantation has reached a plateau of success during the last 5 years 
since the mortality rate is similar for transplantation and maintenance dialysis, es- 
pecially if cadaveric transplants are involved. Koontz notes that "one of the most 
limiting factors in transplantation today is an inadequate supply of cadaveric kidneys". 
In addition, he believes that the patient on dialysis should be informed that he only has 
a 20 percent chance of obtaining a transplant, provided the number of transplants each 
year remains stable. The procedure of choice by a patient, i.e., either dialysis or 
transplantation, will probably be renal transplantation if a good tissue match can be 
obtained. This is because a large number of transplant recipients have returned to a 
relatively active and normal life. In addition, there are still many problems which 
confront the patient on maintenance dialysis. 

Because renal transplantation is the procedure of choice by persons suffering 
from renal insufficiency, organ preservation has become an increasingly important 
problem. This relates not only to the procedures used to obtain and store the kidney 
but also to the time factor needed to sufficiently tissue-type the recipient and donor. 

Belzer (1974) has recently divided renal preservation into four categories. These 
include 

(1) Simple hypothermic storage 

(2) Short- term preservation 

(3) Intermediate-term preservations 

(4) Long-term preservation. 

Simple hypothermic storage has been used extensively and has the chief advantage of 
simplicity and cost. In this storage procedure, the kidney is flushed with a chilled 
solution (usually Collins' solution) to cool the kidney core rapidly. It is then stored 
at 2 to 4 C until it is used. However, its major limitation is its short viable preser- 
vation time, usually 5 to 8 hours. 

Short-term preservation was introduced clinically in 1967 and consists of a con- 
tinuous pulsatile perfusion. As pointed out by Belzer the basic principles of continuous 
perfusion are pulsatile flow, hypothermia, membrane oxygenation, and a perfusate 
containing albumin as well as lipids. The advantage of continuous perfusion is that 
kidneys can be successfully stored up to 3 days, thus permitting the transplantation to 
be performed at the most convenient time for both patient and surgeon. In addition, the 
increased preservation time permits viability testing to be performed before the 
transplantation. 



C-4 

Intermediate and long-term preservation will provide the next technical break- 
throughs in renal transplantation. Only when these occur will it be possible to perform 
the more time-consuming methods of donor- recipient matching {mixed lymphocyte 
culture) and for bank storage of donor kidneys to exist. Long-term survival and in- 
creased percentages of graft acceptance will depend on future development in techniques 
and theory involving donor-specific immunosuppression. 



HISTORICAL ACCOUNT 



The historical account is divided into four sections, i.e., (1) animal transplanta- 
tion, (2) human transplantation, (3) renal preservation, and (4) immunosuppressive 
therapy. For each section, the chronological events are discussed in some detail, 
although each event in itself may not be described in detail. There were certain surgi- 
cal or related events which are important in discussing kidney transplantation and are 
covered either in the animal or human transplantation section. The historiographs 
are intended to illustrate the interrelationship of events from a chronological view. 



Animal Transplantation History 



Kidney transplants were not attempted in animals until the early 1900's. The 
main difficulty in effecting a transplant was overcome by Carrel in 1902 when he devel- 
oped a successsful method of anastomosing blood vessels. He introduced fine round- 
body suture needles threaded either with oiled thin linen or cotton for constructing 
end-to-end or end-to-side arterial and venous anastomoses. Carrel and Guthrie also 
developed the "patching method" for anastomosis of small vessels. Anastomosis by 
this method consisted of extirpating a vessel together with an area or patch from the 
vessels origin, the patch being so cut that the mouth of the extirpated vessel is in the 
center of the patch. The edges of the patch are then fixed to the edges of a suitable 
opening made in the wall of another vessel. 

Ullmann was the first to carry out both autologous and homologous kidney trans- 
plants in dogs. In addition, he reported the first heterotransplant when he made a 
transplant from a dog to a goat. DeCastello did a homologous kidney transplant in a 
dog and noted that the kidney secreted about 1200 cc of urine over a 40-hour period. 
Carrel at the same time also performed several autotransplants on dogs but all trans- 
plants were eventually destroyed by infection. In these early transplants, the kidney 
was anastomosed in the neck region. 

In 1905, Carrel and Guthrie compared the function of a renal homograft trans- 
planted 3 days earlier with a normal kidney. They found that the rate of excretion was 
five times greater than normal, but that metabolite excretion was decreased, i.e., 
urea, organic sulfates, and pigments. In this experiment, the renal artery of the 
transplant was sutured to the carotid artery and the renal vein to the external jugular 
vein while the ureter was anastomosed to the esophagus. That same year, Floresco 
perfornned renal homotransplants in the inguinal area, neck, and renal fossa (lumbar 
area) of dogs. From these studies, he concluded that the renal fossa was the most 
suitable location and the inguinal area the least suitable (Beathard). One year later, 
Carrel and Guthrie reported they had transplanted both kidneys from one dog to another 
by an "in mass" technique. In this study, the kidneys and surrounding tissues were 



C-5 

removed in mass with segments of the aorta and vena cava. These vascular segments 
were then anastomosed in continuity to the aorta and vena cava of the host. The dog 
survived for 10 days and the urine was within normal limits on the eighth day. 

In 1907, Stich reported on a dog kidney transplanted into the iliac region, the 
renal vessels being anastomosed end-to-end to the iliac vessels with the ureter being 
grafted into the bladder. However, the normal kidneys were not removed and the use- 
fulness of the transplanted organ could not be determined (Beathard). A year later, 
Carrel and Langlois attempted renal transplantation in cats. Langlois attempted a 
double transplantation of homologous kidneys into the abdominal cavity of cats while 
Carrel transplanted in mass both kidneys to 14 cats (a contralateral nephrectomy being 
performed immediately). The longest survivals in this series of 14 cats vi'ere 36 days. 
A significant observation of the transplant at autopsy was that plasma cell infiltration 
was more marked in the kidney cortex than in the medulla. All subsequent observers 
were to note infiltration of these cells and their next of kin, the lymphocytes. 

Zaaijer performed the first successful long-term autotransplant in a dog in 1908. 
The kidney was transplanted in the inguinal area and the ureter implanted into the 
bladder. This transplant maintained the animal for 4 years after the removal of the 
contralateral kidney. In 1913, Lobenhoffer attached an autogenous kidney to the splenic 
vessels and removed the other dog kidney. He studied the effects of diuretics (sodium 
chloride, sodium sulfate, and sugar) at various strengths and observed that the trans- 
planted kidney was as responsive to these diuretics as the normal kidney. Lobenhoffer ' s 
studies tended to prove that the urine secreted by an autograft was similar to that 
secreted by a normal kidney (Holloway). 

Quinby followed the above observations in more detail in 1916 when he directed 
his efforts toward determining the relation of the renal nerves to renal function. He 
observed similar effects as noted by Lobenhoffer and suggested that there are no 
secretory nerves to the kidney. Quinby was able to show that homografts acted simi- 
larly to autograft transplants, albeit there might be a delay in urine being produced 
initially. The transplanted kidney would recover with time and secrete similarly to 
a normal kidney. 

Esser in 1917 demonstrated that organs and large flaps of tissue could survive 
on the circulation provided by a single artery and vein. Initially, transfer of tissue 
composites were limited by the length of the supplying vessel. Now the vessels of the 
transplant could be anastomosed to suitable vessels at the site of need (Smith). 

The next work on kidney transplantation was by Dederer in 1920 when he made a 
homologous transplant in puppies in which the recipient and donor in the experiment 
were sisters. The recipient died of distemper on the 25th day after the operation. 
The fact that this successful experiment was made upon members of the same litter 
suggested to him that a biologic phenomenon might be responsible for the failures pre- 
viously attributed to mechanical difficulties. 

In 1926, Ibuka, Williamson, and Holloway all tended to confirm that whereas 
autogenous kidney transplants would maintain the life of the animal for months after 
the other kidney was removed, homologous transplants functioned for only a few days. 
All noted the characteristic plasma cell and lymphocyte infiltration followed by general 
acute nephritis (tubular destruction) and ascending infection. Holloway noted that the 
destructive process appeared to start within 24 hours. Williamson suggested at this 
time that kidney homotransplantation might be justified in the human if (1) a cadaver 



C-6 

kidney was obtainable, (2) the transplant could be completed within 2 hours of the 
donor's death, and (3) the blood groups were matched. However, there is no evidence 
that he attempted such a procedure. 

Wu and Mann followed up the observations noted by the above authors in 1934 
when they made a microscopic study of the day-to-day progress of autologous and homol- 
ogous transplants in the dog. They were able to confirm that monocytic interstitial 
infiltration and tubular necrosis occurred in honnografts during the rejection process. 

Parkinson and Woodworth performed renal transplants in goats of different species 
in 1947. The kidneys were transplanted in the neck using vitallium tubes for the anas- 
tomoses; the longest survival was 10 days. In 1949, Lefebvre performed transplants 
in the neck and again used vitallium prostheses. He showed that a dog homograft 
could clear urea, and he had one dog live for 21 days with a normal blood urea nitrogen 
being maintained for 19 days. 

In 1950, Dempster defined the "toxic syndrome" in animals as consisting of fever, 
anorexia, leucocytosis, and a .oss of weight. Lawler in 1950 discussed the disintegra- 
tion of the homotransplanted kidney and suggested that the cellular infiltration seen 
in humans coincided with the toxic state seen in dogs. Dempster and Simonsen made 
further detailed investigations into the histological appearance of rejection in animals 
in 1953. They noted that round cell infiltration was more pronounced in the cortex 
than the medulla. In addition, they noted that many of these cells represented plasma 
cells, the small intrarenal arteries occasionally exhibited endothelial swelling, and 
there was perivascular infiltration. 

Graves, in 1954, studied the distribution of the intrarenal arteries and showed 
that irrespective of the distribution pattern of the extrinsic arteries, each artery that 
entered the kidney was an end artery having its own segmented distribution within the 
kidney. Reis and Esentler in 1959 followed up on Graves' work and found that the 
frequency of multiple renal arteries in 500 cadavers was approximately 19 percent in 
the left kidney and approximately 15 percent in the right kidney, while the frequency 
of multiple renal veins was less than 1 percent on the left side and almost 11 percent 
on the right side. Thus, based upon these studies, the left kidney of the donor is pre- 
ferred due to few renal anomalies, the greater length of the left renal vein, and the 
more optimal anatomical relationships of the iliac vessels (Smith). 

In 1960, Mannick et al. performed renal transplants in dogs with experimentally 
induced uremia. They were able to show that renal transplants functioned two to three 
times longer than transplants carried out in normal dogs (renal autografts functioned 
for a mean of 19.2 days in the uremic dog and only 5.9 days in normal dogs) (Calne, 
1963). 

Zukoski, in 1961, reported on reciprocal renal transplantation in two boxer dogs 
not known to be related. In the first-set transplants, function was maintained for 39 
and 60 days, respectively, while a second-set transplant faired even better, producing 
urine for 88 days. These studies showed that although unmodified canine renal homo- 
grafts usually cease functioning after 5 to 8 days, there can be exceptions. In fact, 
Koo et al. have had a single dog go for 123 days. However, in these studies, the dogs 
have undergone at least one episode of acute rejection which commences 13 to 19 days 
after transplantation (Porter). 



C-7 
Human Transplantation History 



The first known attempt to combat acute uremia occurred in 1902 when UUmann 
anastomosed a pig renal graft to the circulatory system of a patient, but it failed for 
technical reasons. In 1905, the first renal heterotrans plant occurred when Princeteau 
inserted slices of rabbit kidney into the kidney of a child suffering from renal insuffi- 
ciency. He claimed the immediate results were beneficial, but the child died on the 
I6th day. The following year, Jaboulay anastomosed kidneys from a pig and goat, re- 
spectively, to the artery and vein of two patients. Diuresis was obtained for approxi- 
mately I hour, but the grafts soon ceased to function and were removed a few days 
later. Graft failure was attributed to vascular thrombosis (Reemtsma et al. , 1964). 

The next attempt at heterotransplantation in the human occurred in 1910 when 
Unger took both kidneys from a monkey and placed them in the groin of a uremic 
patient. The transplant did not function. The patient died 32 hours after transplanta- 
tion and autopsy again showed venous thrombosis. The procedure represented an 
advance since the intracorporeal location was used for the first time. In addition, the 
desirability of genetic similarity between donor and recipient was recognized. 

In 1914, an event occurred which would later influence kidney transplantation 
directly and this was the introduction of the first "artificial kidney" by Abel et al. 
They dialyzed the blood of experimental animals through celluloid tubes and used the 
anticoagulant hirudin. Although they realized the potentials of this concept, it re- 
mained for Kolff to pioneer the construction of an artificial kidney at a much later date. 

Another attempt at heterotransplantation in the human occurred in 1923 when a 
patient suffering from mercury bichloride poisoning was transplanted with a lamb 
kidney. However, this patient also died 9 days later. The next human application did 
not occur until 1936, but it was a very decisive event in that Voronoy, a Russian sur- 
geon, reported the first use of a renal transplant from a human donor. The kidney 
was obtained from a recently deceased person and was anastomosed to the femoral 
blood vessels of the recipient who was suffering from mercury poisoning. The graft 
produced small amounts of urine for 2 days, but the patient died on the third day 
(Groth). The first report of a human donor in the United States occurred in 1945 when 
Landsteiner and Hufnagel transplanted a kidney from a cadaver to the brachial artery 
and cephalic vein of a young woman in acute renal failure. The patient's own kidneys 
began to function a few hours later and the transplant was removed 48 hours later. 
However, the transplant did not secrete any significant amount of urine during this 
period (Hume). 

In 1943-1947, Kolff pioneered in the construction of dialyzing apparatus which 
proved successful in the therapy of uremia in man. This apparatus consisted of long 
cellophane tubing, wound spirally about a large drum, which revolved horizontally in 
a dialysate bath. Blood flow was from the radial artery to the cellophane tubing with 
return to the antecubital vein being accomplished by a roller pump. 

The first description of clinical renal transplantation in an intra-abdominal loca- 
tion occurred when Lawler et al. transplanted a cadaver kidney to a woman suffering 
from polycystic disease in 1951. One of the polycystic kidneys was removed and the 
transplant placed in the renal fossa with anatomic reconstruction of blood vessels and 
ureter. Approximately 2 months following transplantation, the authors injected indigo 
carmine dye and noted that the dye emerged from both ureters, but in poorer 



C-8 

concentration from the transplanted side. This was a poor test for a transplanted kidney 
since the remaining polycystic kidney was capable of sustaining life. 

No fewer than eight attempts at transplantation occurred in France in 1951. One 
was reported by Servelle, two by Dubost, and five by Kuss. In each case, the trans- 
plant was placed in the iliac fossa and the renal vessels were anastomosed to the iliac 
artery and vein. Urinary drainage was accomplished via a cutaneous ureterostomy. 
The series by Kuss et al. was the first in which grafts from living donors were em- 
ployed. The kidneys were obtained from patients suffering from urological diseases 
and thus constituted free organs. The longest implant in this series lasted 3-1/2 
months but when the kidney was removed, it was found to be secreting only a few drops 
of urine. Most of the other patients in the French series Lived from 16 to 20 days 
(Groth). 

In 1952-53, the first successful renal transplants were reported from Peter Bent 
Brigham Hospital in Boston. The significance of this series was that this was the first 
time that a renal transplant would sustain life for any length of time (6 months). The 
first of the patients from this series was operated upon by Scola in the nearby town of 
Springfield. The transplant was a free kidney and was anastomosed to the splenic 
blood vessels. Only slight diuresis followed and the patient was returned to Peter 
Bent Brigham Hospital shortly before death. By 1955, eight more renal transplanta- 
tions were performed by Hume and his colleagues using either free or cadaveric kid- 
neys. The renal blood vessels were anastomosed to the femoral artery and vein with 
the kidney being placed in the upper part of the thigh. Only the last transplant produced 
ample amounts of urine for 6 months, but of the first six cases, three functioned for 
more than a month. Use of the artificial kidney was made during this series when all 
the transplants went anuric for 8 to 19 days before starting to function. This series 
was to herald the advances to be made during the modern era of kidney transplantation. 

The first renal transplant in which the donor was a healthy relative occurred in 
1953 when Michon performed a mother-to-son kidney transplant. The boy had his only 
kidney removed when he suffered a traumatic hennorrhage. Seven days later a kidney 
was transplanted from his mother who matched his blood group. The transplant func- 
tioned immediately without the period of acute tubular necrosis seen in cadaver trans- 
plants. However, the transplant functioned only 22 days after which the patient became 
uremic and died 10 days later (Beathard). 

The only situation in which there presumably would be no immune defense mecha- 
nism would be if identical twins could be transplanted. In 1954, Merrill and his group 
performed the first renal transplantation between monozygotic twins. The kidney was 
placed retroperitoneally and anastomosed to the iliac blood vessels while the ureter 
was implanted in the bladder. The patient survived for 8 years before dying of a 
myocardial infarction. In 1959, the first renal transplants between nonidentical twins 
were reported by Hamburger in France and Merrill in the United States. All three 
cases received sublethal total- body irradiation and were treated with cortisone prior 
to transplantation. Two of the kidneys were still functioning 2-1/2 years later while 
the third case died on the I2th day from the effects of 600 r of total- body irradiation. 
Murray and his group abandoned total-body irradiation because of repeated failure, and 
in 1962, began to cautiously use immunosuppressive drug therapy and recorded the first 
instance of reversing a rejection reaction via drug treatment. This was to be an ex- 
tremely pertinent observation because it indicated that the immunological rejection pro- 
cess was not an "all-or-none" phenomenon but could be treated by proper and timely 
therapy. 



C-9 

Although Carrel had perfected his technique of anastomosing blood vessels, over 
half a century elapsed before Shackman and Dempster (1963) and Hardy (1963) per- 
formed an autotransplant in man. Shackman and Dempster treated a patient suffering 
from renal artery stenosis by removing the kidney, excising the stenosed arterial seg- 
ment, and retransplanting the organ back in the iliac fossa. Hardy salvaged a kidney 
via autotransplantation when the ureteroureteral anastomosis for repair of a high ureteral 
injury resulted in stricuture (Porter). 

Heterotransplantation was forgotten over a 40-year period because of the feeling 
that investigation along these lines had insurmountable problems. Reemtsman (1963) 
and Hitchcock in 1963 reexamined the possibility that heterograft function might be sus- 
tained with the aid of various immunosuppressive agents. Both of these investigators 
demonstrated that immediate urine excretion was possible using the chimpanzee, rhe- 
sus monkey, or baboon. In fact, Reemtsma had one patient with paired chimpanzee 
heterografts function normally for 22 weeks after the operation. 

Although renal transplantation was initially attempted in adults, Cerilli demon- 
strated in 1970 that an infant 9 months old could be successfully transplanted. The 
patient has survived with good function for over 6 years. In addition, transplants have 
now been carried out on infants 2 and 4 months old without technical difficulty. How- 
ever, one infant rejected the transplant 3 months later while the other died on the 
second posttransplant day (Najarian). 



Renal Preservation History 



As early as 1908, Carrel noted that the blood supply of the dog transplanted kid- 
ney could be stopped for approximately 1 hour without interfering with its subsequent 
function. In addition, he noted that the kidney could be perfused to its advantage with 
Locke's solution or serum prior to transplantation. It was at this time that he sug- 
gested perfusion as a means of renal preservation, although the perfusions did not 
exceed 50 minutes. Then, in 1924, Avramovici performed a unilateral homotransplan- 
tation in loco on a dog in which the donor kidney had been preserved in the cold (icebox) 
for 8 hours. I'he dog's contralateral kidney was removed 2 weeks later and the animal 
lived for an additional 36 days. He also studied renal preservations in the cold for 
periods as long as 30 hours, but none was as successful as the 8-hour preservation. 
Oudot attempted to extend Avramovici's record of 8 hours by keeping the transplants 
cooled to 4 C for periods up to 8 days. He atterapted this in 1948 in two dogs, but in 
both cases, infarction of the kidneys occurred immediately after transplantation (Hume). 

In 1951, Lefebvre studied the effect of perfusing the dog kidney and storing it at 
low temperatures prior to transplantation. He was able to demonstrate that kidneys 
stored up to 24 hours were capable of functioning after transplantation, as measured 
by diuresis, chloride output, return of oxygen consumption, excretion of bilirubin and 
urochrome, and urea output. However, all of the indices except chloride output were 
below that of a normal functioning kidney. At this same time, Dempster suggested 
that perfusion was probably the best modality of preservation for solid organs. Initially, 
he met with repeated failures in attempts to successfully store dog kidneys for 6 hours 
by hypothermia. He achieved success only when a suitable perfusate was used, red 
blood cells were removed from the organ, and the interval between kidney removal and 
cold storage was shortened. 



C-10 

In 1953, Lovelock proposed the hypothesis that freezing produces extracellular 
ice crystallization and that these crystals are formed at the expense of the intracellular 
water. As a result, the cells die of dehydration ("hypertonic cellular damage"). This 
theory was of importance since at C the barrier of freezing damage has to be 
overcome. 

A variety of cooling techniques were expounded when hypothermia was first pro- 
posed as an adjunct to cardiac surgery. Among these was an idea by Kjoellgren in 
1954 which proposed intra-abdominal cooling by peritoneal lavage. The advantage this 
method offers is a rapidly induced hypothermia and a reduction in warm ischemia time 
prior to the kidney being excised and perfused. This would be of value especially in 
cadaver transplants. 

In 1957, Mitchell and Woodruff demonstrated that by cooling a kidney, one could 
significantly increase the period of ischemia the organ would tolerate before permanent 
damage occurred. Although the protective effects of hypothermia had been recognized, 
it was Levy in 1959 who pointed out that the oxygen consumption of the kidney decreased 
exponentially with decreasing temperature. He noted that when a canine kidney was 
cooled to 30 C internally, the oxygen consumption was roughly 43 percent of the normal 
value. At lower renal core temperatures (4 to 5 C), the oxygen consumption was only 
5 percent of normal. This was to be further substantiation for the use of hypothermia 
since it depressed kidney metabolism. 

Lovelock and Bishop reported the first use of dimethyl sulfoxide as an endocellular 
cryophylactic agent in 1959. They showed that the dinaethyl sulfoxide permitted a re- 
duction of the temperature in stored kidneys to subzero levels without freezing. Fol- 
lowing reimplantation of the kidney (autograft) in the dog, the kidney functioned and 
secreted urine, but no long-term survivals were achieved. The significance of this 
event may eventually be of importance for long-term storage of organs and the develop- 
ment of cyroprotective agents which might prevent problems of freezing and thawing. 

In I960, Lapchinsky reported successful renal storage when he extracorporally 
perfused dog kidneys utilizing constant perfusion. In that study, he pumped cold blood 
through the kidneys for approximately 1 hour and then stored them at 2 to 4 C for 26 to 
28 hours. Prior to autografting, he then pumped warm blood through the organ for 1 
hour. In some of the experiments, a satisfactory return of renal function was noted 
with immediate removal of the undisturbed kidney. However, in most of the studies, 
the contralateral kidney was usually not removed until 2 months later, but eight dogs 
lived over 3 years after such a procedure. 

Earlier, Warburg (1927) and Levy (1959) had demonstrated the relationship of 
oxygen consumption to kidney cortex slices and the effect of temperature on the metab- 
olism of the whole organ, respectively. In I960, Semb and associates showed that 
reducing the metabolism of the kidney by cooling enabled it to withstand relatively pro- 
longed periods of ischemia. In addition, they confirmed Levy's work and showed that 
oxygen consumption decreases exponentially with falling temperatures, reaching a very 
low level at temperatures within a degree of zero. 

A significant event occurred in 1963 when Calne performed the first successful 
12-hour storage with an immediate contralateral nephrectomy. At the same time, 
Humphries showed that storage could be increased to 24 hours followed by an immediate 
contralateral nephrectomy. In prior studies to those two events, the contralateral kid- 
ney was retained for a period of time before it was removed. The principal changes in 



C-11 

Humphries' method included the use of whole blood in the perfusate, slightly higher 
perfusion pressure, a glass wool filter, and a membrane lung. He believed use of 
blood in the perfusate to be the most beneficial factor, while the glass wool removed 
aggregates of altered platelets and leukocytes and the membrane lung was chosen be- 
cause it damaged the blood less. 

Other events during the same year (1963) were the demonstration by Knight and 
associates that if the dog kidney is cooled by arterial perfusion at 4 C and 140 mm Hg 
pressure, one can successfully preserve renal function after 6 hours of extracorporeal 
storage at this temperature (Guthrie et al.), and the demonstration by Calne and asso- 
ciates that simple surface cooling with ice will considerably minimize the ischemic 
damage in stored dog kidneys. In the latter study, removed kidneys were placed in a 
polyethylene bag surrounded by ice. These studies confirmed that this method would 
permit adequate function in the reimplanted kidney after up to 12 hours of preservation. 
They also noted that if the storage time was extended to more than 17 hours, severe 
irreversible damage occurred in the kidneys and useful function was not obtained, 

Keeler demonstrated the value of using intracellular electrolyte solutions to cool 
and flush out blood from kidneys prior to long-term storage in 1966. They perfused 
dog kidneys with either a solution rich in potassium and magnesium ions (intended to 
simulate intracellular fluid) or Tyrode solution. They were able to show that kidneys 
perfused with the former solution gave adequate kidney function, whereas a kidney 
perfused with cold Tyrode solution failed completely. It was only by use of this modi- 
fied intracellular fluid that results superior to surface cooling were obtained. 

The demonstration in 1967 by Belzer and associates that dog kidneys could be 
preserved for up to 72 hours produced a new wave in perfusion technology. The basics 
of this perfusion technique included pulsatile flow, hypothermia in the range of 6 to 12 C, 
membrane oxygenation, and usually cyroprecipitated plasma as the perfusate. This 
perfusion technique became the standard method of cadaver-kidney preservation when 
it was demonstrated that it could also preserve cadaver kidneys for up to 72 hours' 
storage. 

In 1969, Collins developed a solution similar to that proposed by Keeler except 
that it contained high concentrations of potassium and low concentrations of sodium. 
He demonstrated that kidneys flushed and cooled with this intracellular solution could 
be shipped for long distances and function satisfactorily in most instances. 

A limiting factor in pulsatile perfusion was the gradual increase in peripheral 
vascular resistance associated with decreased flow, parenchymal edema, and poor 
function after transplantation. Starling and associates in 1972 showed a straight-line 
relationship between lysosomal enzyme concentrations in the perfusate and an increase 
in peripheral vascular resistance. In addition, they demonstrated that methylpredni- 
solone could stabilize the lysosomal membrane, retard the release of acid hydrolases, 
and thereby delay the appearance of increased vascular resistance. 

Long-term preservation awaits a breakthrough but Dietzman has recently (1973) 
"supercooled" a dog kidney to -22 C which gave life-supporting function after autotrans- 
plantation. The only drawback was that the entire cooling (1 to 2 C per minute) and 
rewarming period had to be completed within 15 minutes to produce viable kidneys. 
Another recent event has been the development of a new perfusate by Sacks et al. of 
modified intracellular electrolyte composition made hyperosmolar with mannitol. The 
first instance of 72-hour canine kidney preservation has been accomplished following 



C-12 

initial perfusion and hypothermic storage with this solution. In addition, kidneys have 
been stored successfully for 48 hours in which 30 minutes of warm ischemia was 
followed by flushing the kidney with 200 cc of the above solution for 5 minutes and then 
maintaining it in the same solution at 2 C. 



Immunosuppressive Therapy History 



The current approach to immunosuppression following human renal transplanta- 
tion involves combination therapy using pharmcological and biological agents and/or 
surgical techniques. The most widely accepted standard protocol involves the use of 
azothioprine, antilymphocyte globulin (ALG), and prednisolone (or prednisone) admin- 
istered at various times and in varying dosages following transplantation. 

The development of all of the currently accepted methods of immunosuppression 
employed in renal transplantation followed a similar developmental pattern. The 
effects of these agents on humoral antibody production was noted initially followed by 
studies of the effects of the various immunosuppressants on the cell-mediated responses. 
This closely parallels the development of the theories of immunologic function, in that 
the earliest known function of the immune response to be identified and studied was the 
humoral and not the cellular responses. However, in the mid-1940's Gibson and 
Medawar clearly focused the attention of transplantation researchers on the fact that 
the transplantation rejection phenomenon was due primarily to a cell-mediated immune 
response and that the rejection phenomenon was donor specific. Transplantation re- 
searchers soon realized that agents which immunosuppress the transplant recipient 
could prolong the survival of organ grafts, and in the 1950's widespread experimenta- 
tion began, using various approaches to immunosuppression for the prolongation of 
skin grafts and renal transplants first in animals and subsequently in man. 

The five major approaches to immunosuppression in renal transplant recipients 
included purine analogs, steroids, radiation, ALG, and others {including surgical or 
mechanical lymphocyte depletion and other classes of chennical agents). Two very 
early findings by Hektoen and Corper ( 1921) and Hektoen (1915) 'demonstrated that 
mustard gas and X-irradiation, respectively, in rabbits reduced the antibody response 
to soluable antigens. 

Despite these early observations, the concepts of immunosuppression were not 
applied for approximately 30 years due to the lack of correlation between the theories 
of imnnunological function and the clinical observations involving organ rejection. The 
elucidation of two major theoretical concepts was required for the development of 
effective immunosuppressive therapy. These were 

(1) The genetic histocompatiblity basis of organ and tissue 
transplantation 

(2) The role of delayed hypersensitivity (small lymphocytes) and 
the second-set rejection phenomenon in transplantation. 

Most immunologists recognize the work of Little and Tyzzer in 1916 as the initial ob- 
servation involving the genetic basis of the acceptance or rejection of spontaneous 
tumors in various inbred strains of mice. In 1914, Murphy, using histopathological 
means, implicated the small lymphocyte in the rejection of skin transplants. Ten years 



C-13 

later, Holman (1924) hypothesized the presence of an active immune mechanism in the 
rejection process when he observed that an animal which had rejected a first graft 
would reject a second graft much more rapidly. 

The application of immunosuppressive therapy to renal transplantation involved 
a key series of experiments in the mid to late 1940's. These began with the experi- 
ments of Gibson and Medawar in 1943 which described the time requirement for second- 
set rejection of skin grafts, when the grafts were from the same donor. In the following 
year, Medawar concluded that the accelerated rejection consistently observed in second- 
set graft rejection was donor specific. However, perhaps the most significant theoreti- 
cal finding in terms of eventual clinical application was reported by Medawar in 1945. 
Medawar determined that the rejection of skin transplants in rabbits was the result of 
a systemic immunological reaction and not the result of a local reaction as had been 
previously believed. In 1948 Medawar further supported this hypothesis by demon- 
strating that skin grafts which were placed in vascularized areas of the rabbit's body 
were rejected, while those grafted onto the eye of the same rabbit, a nonvascularized, 
"immunologically-privileged" area, were not rejected. 

The elucidation of the systemic nature of transplant rejection initiated an active 
search for methods of systemic immunosuppression which could eventually be applied 
to the renal transplantation problem. 

In 1950, Germuth and Ottinger deraonstrated that steroids (cortisone) inhibited 
the arthus reactions in rabbits previously sensitized with human serum albumin. That 
same year Dempster applied whole- body irradiation as an immunosuppressant and 
found it useful in prolonging the homograft survival of skin transplants in rabbits. The 
following year (1951), Billingham, Krohn, and Medawar followed up Medawar's earlier 
experimentation and determined that high doses of cortisone could prolong skin grafts 
in rabbits. In 1951, Woodruff, Formon, and Fraser first suggested that an antilympho- 
cyte serum might be useful in abrogating the rejection phenomenon. 

The first direct application of immunosuppressive therapy specifically to renal 
transplantation was performed by Baker et al. in 1952. This approach involved a 
multiple-treatment regimen which included nitrogen mustard, cortisone, and splenec- 
tomy. A year later Dempster performed the first successful canine renal transplants 
using whole-body irradiation as the mode of immunosuppression. In 1959, immuno- 
suppressive therapy was used for the first time in human renal transplant recipients. 
Hamburger perfected the use of whole- body irradiation for the immunosuppression of 
human renal transplant recipients and demonstrated significant prolongation of renal 
grafts. 

The use of whole-body irradiation was considered a high-risk procedure because 
of the high incidence of lethal systemic bacterial infections in irradiated individuals. 
Preceding 1959, the only other agents available, namely steroids and nitrogen mustard 
derivatives, had shown only very limited effectiveness in inducing immunosuppression. 
In 1959, however, Schwartz and Dameshek, utilizing the recently synthesized purine 
analog, 6-mercaptopurine, demonstrated the effectiveness and relative safety of this 
compound in suppressing the antibody response of rabbits to serum albumin. This 
initiated a revolution in the clinical feasibility of large-scale immunosuppression. In 
I960, Calne reported marked prolongation of renal homografts in dogs using 6-mercapto- 
purine. This work was confirmed in the same year by Zukoski, Lee, and Hume. The 
following year Elion et al. working at Burroughs Wellcome and Company, Inc. in 
England synthesized another related compound, namely, azothioprine. In 1961, Calne 



C-14 

elucidated its superior effectiveness in prolonging renal homografts in dogs using the 
azothioprine supplied by Burroughs Wellcome. In the same year Kuss et al. applied 
6-mercaptopurine in humans as adjunctive therapy during renal crisis with some suc- 
cess. Murray et al. in 1962 used either 6-mercaptopurine or azothioprine as the only 
form of therapy in newly transplanted patients with excellent success. Among these 
patients were the first cadaveric transplants to survive more than 12 months. It was 
also discovered in 1962 by Goodwin, Mims, and Kaufman that the steroids could pro- 
duce almost total reversal of rejection crisis and were best used as adjunctive and not 
primary therapy. 

In 1961, Waksman, Arbouys, and Arnason concluded that antilymphocyte serum 
(ALS) could successfully prolong skin grafts in guinea pigs presumably by reducing the 
cell-mediated imm.une response. Two years later (1963), Woodruff and Anderson 
detailed the mechanism of action of ALS in prolonging skin homografts in rats, using 
lymphocytes obtained from individual rats by thoracic duct drainage as a source of 
lymphocyte antigen. During the same year Hume et al. applied high-dose radiation 
directly to the area of the renal transplant in canines and combined this approach with 
chemical immunosuppression. Kauffman et al. applied the technique of local renal 
graft irradiation to human renal transplants in 1965, but found that the advantage gained 
in avoiding systemic infection was outweighed by the radiation- induced nephritis which 
occurred in many cases. 

In 1966, the three major methods of immunosuppression were combined in a 
standard therapy protocol by a group of researchers under the direction of T. E. Starzl. 
In 1963, Starzl, Marchioro, and Waddell completed the first series of truly successful 
clinical renal transplants in which the majority of the eight patients survived 2 or more 
years on a therapy regimen of azothioprine and high-dose steroids employed for rever- 
sal of the rejection crisis. Subsequently, in 1966, Starzl et al. added the more purified 
ALG to the therapy regimen, thus completing the basic regimen that has been used (with 
some minor modifications) to treat patients following renal transplantation until the 
present time. 

The other major line of research that runs parallel to the development of immuno- 
suppressive therapy consists mainly of the areas of immunogenetics and antigen recog- 
nition. The landmark findings in these areas are represented as a parallel column on 
the historiograph entitled "Advances in Transplantation Immunology". Since the 
theoretical consideration of cell surface antigens by Ehrlich in 1900, the major themes 
of these areas of research have been the discovery and categorization of transplantation 
antigens and the interactive role of these antigens in the development of immunologic 
mediated graft rejection tolerance to homo-, alio-, and xenografts. 

The experiments of Little and Tyzzer in 1916 showed that spontaneous tumors 
from specific inbred strains of mice were rejected by other inbred strains of mice, 
thereby demonstrating the genetic basis of the rejection phenomenon. In 1948, Gorer, 
Lyman, and Shell isolated the first transplantation antigen in mice, namely the H-2 
locus. Six years later Dausset described the first human leukocyte antigen which he 
termed the "Mac" antigen. This was followed by successive discoveries in murine and 
human immunogenetics that further defined histocompatibility antigens and related the 
function of these antigens to renal homografts survival. Terasaki was the first to apply 
histocompatibility testing to selected donors. After the first reported transplantation 
between identical twins by Merrill et al. in 1956 and the subsequent renal transplanta- 
tions between twins by Murray et al. in 1958, it became evident that genetic matching 
was an important factor in the prolongation of grafts in the absence of immunosuppres- 
sion. The Torino Workshop on Histocompatibility antigens which was held in 1967 



C-15 

correlated many separate histocompatibility nomenclatures into a standard system that 
could be applied on the clinical level. In the years following 1967 to the present time 
the necessity to tissue type prior to transplantation is still a controversial subject. 
Indeed transplants between twins and other siblings have been highly successful but 
Terasaki in 1974 has shown that more common homograft and cadaveric transplant 
recipients, when treated with the present regimens of combined general immunosup- 
pression, have a graft survival rate of approximately 50 percent regardless of the 
degree of HL-A match or mismatch. 



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C-16 



Hume, D. M., Transplant. Proceed., 4 (4), 625 (1972). 

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Kountz, S. L., Transplant. Proceed., 6(4), 99(1974). 

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Lawler, R. H. , et al. , JAMA, _li7 (1), 45 (1951). 

Lefebvre, L. , Arch. Internat. de Physiol., ^07 (1), 110 (1949). 

Levy, M. N., Amer. J. Physiol., ^97, 1111 (1959). 

Lovelock, J. E., Biochem. Biophys. Acta, 10_, 414(1953). 

Lovelock, J. E., and Bishop, M.W.H., Nature, J_83, 1394(1959). 

Merrill, J. P., Sci. Amer., 201 (4), 57 (1959). 

Merrill, J. P., et al. , JAMA, ^85 (5), 347 (1963). 

Merrill, J. P., et al. , JAMA, J^O (4), 277(1956). 

Murray, G. , and Holden, R. , Amer. J. Surg., _87, 508(1954). 

Najarian, J. S. , et al. , Annals of Surg. , r74 (4), 583 (1971). 

Parkinson, D., and Woodworth, H. C., Exp. Med. Surg., 5, 49(1947). 

Porter, K. A., Internat. Rev. Exp. Path., JJ^, 73 (1972). 

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Reemtsma, K., et al. , Science, 143 , 700(1963). 

Reis, R. H., andEsenther, G. , Amer. J. Anat., 104 , 295(1959). 

Sacks, S. A., et al. , Amer. J. Surg., _1_28, 402 (1974). 

Simonsen, M. , Acta Path, et Microbiol. Scand. , 32, 36(1953). 

Simonsen, M., and Sorensen, F. , Acta Chir. Scand., 99, 61 (1949). 

Smith, J. W., Plast. & Recon. Surg., 3_7 (3), 227 (1966). 

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Unger, E., Klin. Wschr,, 2, 1057(1909), 



C-17 

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C-18 
REFERENCES (IMMUNOSUPPRESSION) 



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Amos, D. B., Transplant. Proc, 3, 71 (1971). 

Bach, F. H., and Amos, D. B. , Science, 156 , 1506 (1967). 

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Bain, B., Vas, M. R. , and Lowenstein, L. , Blood, 2^, 108(1964). 

Baker, R. , Gordon, R. , Huffer, J., and Miller, G. H. , Arch Surg., ^, 702 (1952). 

Billingham, R. E., Krohn, P. L,. , and Medawar, P. B., Brit. Med. J., J., 1157 
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Billingham, R. E., Ferrigan, L. W. , and Silvers, W. K., Science, J_32, 1488(1960). 

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Calne, R. Y., Lancet, I, 417 (I960). 

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Curtoni, E. S., Mattiuz, P. L. , Tosi, R. M., I. The Workshop Data. Histocompati- 
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Dausset, J., Ivanyi, P., and Ivanyi, D., Series Haemat. , 11, 51 (1965). 

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C-19 

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C-20 



Woodruff, M.F.A., and Anderson, N. F. , Nature (JLond.), _200, 702 (1963). 
Zukoski, C. F., Lee, H, K., and Hume, D. M., S. Forum, jj,, 470 (I960). 



ANIMAL TRANSPLANTATION 



HUMAN TRANSPLANTATION 



RENAL PRESERVATION 



1902 Ullman First known attempt to combat 
acute uremia by anastomosing a 
renal graft to the circulatory 
system of a patient 

MR/D/W/ / 



1906 Jaboulav First s 



WIMAL TRANSPLANTATION 



HUMAN THANSPLANTATION 



RENAL PRESERVATION 




MH/D/U/P.GW 



mb/s/u.w/p.g/m; 



COOES 

NFI Nonmision-Oilo 
MR MLDKin-OiwIM 



i 



Mtusmif.am 



HISTORIOGRAPH - KIDNEY TRANSPLANT 



ADVANCE 



ADVANCES IN TRANSPLANTATION 
IMMUNOLOGY 



IMMUNOSUPPRESSIVE THERAPY 
Anti-lymphocyte Serum 



Isolstod litii hliiocornpiillbllliv 
oeui (H-II In mice 

MniOMICIM 



ieS4 Dauiuii Oancilon ol rhs <liii laukocvD 
sntiBf n (Mac Aniinonl 
MR/D/W/G/M 



1963 Sliiil Dimoniii 



MR/5/UfG/M 



onunoiogmli) uiubu o' 



MR/SAVfG/M 



MR/SMfG/M 

19T0 P»«»fU>n LVWKlKVt" ''Om 4l»in 



CLINICAL OBSERVATIONS SUPPORTING THE 
IMMUNOLOGIC BASIS OF GRAFT REJECTION 



— 1915 HikiMn X-ray la 



Purine Amalogues 



- 19:4 Hotmin OIkikvi 



- 1943 Gibion Otia 



MR/D/W/G/M 



■ 1944 MedaMF Cd"C 



MR/DrtN/G/M 



- 1S4& Medawor Con 



- 1948 MeaawK Olm< 



MR/S/W/P/G/M 



MR/S/DM/G/M 



traj-ti(tlbr>t betiovvd Hki 
.d 

MR/S/U/G/M 



MR/OAJ/G/P/M 



logically prMlagid iltii 

MR/S/U/P/M 



> Oamot'^gh &fnvrcaptopuri 



19S1 Ellon Svnihailiol 



DCV/D/U/P/C 
jt 6-MP .n humsni 1 



1962 Muf'iy G MP Bi 






- 1961 BilKnghim High dou conlioni piolongi 19SI t 

ikin (ila^nli tuivfvil 
MR/D/W/P/M 



- 1962 itVet Supttlor stlect ol 



- 1953 Otminin Ktdniy nomittniijiin 



- 1969 Himburgti Sue 



MR/DAV/G/M 



1961 Wlktman Prolongtd Uin gialu in gi 



-1902 GiKidwin Firti iucc«Blul ub of 

imoidi In Wdnay utniplmi 
ptilenli (ffvefal o( nJ«citon 



(irtnlva ilaix 

MR/D/U/G/P/M 



1963 tVwHlruK DnoiM 



>n ol ALS In iMtongaiifl 
MR/S/W/C/M 



CODES 

NR Nonmusion-OtiBnied Retoj'ch 

MR Mlsion'Odenied Reiaarch 

OV Oeveloomeni 

S Sigmliuni Event 

D Oec'Sivs Event 

U US Event 

W Foieign Event 

G GoverruYMnt Funding 

P Ptfvate Furtdifig 

M Medlul School - HoiplttI - 
Univtnttv 

GL Goiwnmtni L^bofitorv 

C ComMnv 



rul WH m ALS in a 
MR/SAV/G/M 



HISTORIOGRAPH - KIDNEY TRANSPLANT (IMMUNOSUPPRESSION) 



D-1 
APPENDIX D. ORAL CONTRACEPTIVES 

INTRODUCTION AND SUMMARY 



Oral contraceptives have achieved rapid and widespread acceptance. The virtually 
100 percent effectiveness of this regimen as well as the intense interest in combating 
the spiraling population growth were prime factors in its acceptance by more than 50 
million women, including 10 million in the U. S. (George Washington University Medi- 
cal Center, 1974). The dollar value of the market for oral contraceptives is nearly 
$200 million and growing. Varying compositions of progestin alone or in combination 
with estrogen are used. Approximately 30 varieties of oral contraceptives are cur- 
rently manufactured. 

Oral contraceptives were first suggested in 1921 by Haberlandt as a speculation 
resulting from effects of transplanting whole organs from pregnant to nonpregnant ani- 
mals. They were suggested again in the mid- 1930's by Kurzrok, folio-wing discoveries 
by Makepeace and others on the effect of progesterone and estrogens in ovulation, and 
in 1945 by Albright, who attempted to correct endocrinal disorders with oral prepara- 
tions of estrogen and progesterone (PetrOTW, 1966). None of these suggestions led to 
innovative development because they came at a time when the state of the art was still 
inadequate to supply the necessary technology. Work actually began in 1951 when 
Pincus, at the request of a philanthropist, began studying the possibility of controlling 
fertility by chemical nneans, although he did not synthesize or discover oral contracep- 
tives, as is commonly supposed (Drill, 1976). 

One of the striking features of this case is the complex, truly international inter- 
play of the channels of development. The work encountered resistance from anticon- 
traception factions in society and from drug companies unwilling to accept the risk that 
marketing the product nnight have a detrimental effect on their other commercial activ- 
ities. Thus, management decisions to proceed were crucial, and early testing was car- 
ried out in Puerto Rico and Mexico by U. S. companies. Official U. S. Food and Drug 
Administration (FDA) approval for Enovid®was finally given in 1959. 

Three regimens are currently used: ( 1) combined (introduced in 1960), (2) sequen- 
tial (1963), and (3) continuous progestin (minipill) (1972). In the combined regimen, 
used most often, progestin and an estrogen are used together in a tablet and are admin- 
istered daily for 20 or 21 days. In the sequential regimen, an estrogen is administered 
for 14 to 16 days followed by the daily administration of the combined estrogen and 
progestin tablet for 5 or 6 days. The continuous progestin regimen, used least often 
uses progestin alone. The historical development and potential side effects of these 
oral contraceptives are discussed in the next section. 



HISTORICAL ACCOUNT 



The initial concept of an orally administered contraceptive chemical was suggested 
by Haberlandt in 1921. Having successfully induced temporary sterility in rabbits by 
transplanting ovaries from pregnant animals into nonpregnant animals, he suggested 
that extracts made from the ovaries of pregnant animals be used as oral contraceptives. 



D-2 

Haberlandt (1924) then showed that extracts of corpora lutea from pregnant cows ren- 
dered rabbits infertile. In 1927, he reported that ovarian extracts given orally to mice 
produced temporary sterility. 

In 1921, Fellner reported that lipid extracts of ovarian tissue of pregnant cows 
had potent biological effects. Through 1928, Fellner continued to report experiments 
showing the estrogenic properties of his ovarian extract. In 1923, Allen and Doisy de- 
veloped a convenient assay for estrogenic activity. 

The discovery that urine from pregnant women contained substantial concentra- 
tions of estrogenic hormones was fortuitous, for it provided a readily available source 
of these hormones. The isolation of the hormone responsible for estrus was reported 
by Doisy in 1929. In 1930, Doisy and Butenandt independently determined the structure 
of estrone. Almost simultaneously, Marrian (1930) isolated estriol from pregnancy 
urine. 

In 1929, Corner and Allen developed a convenient bioassay for progestational ac- 
tivity. This test facilitated the isolation of progesterone from the corpus luteum. By 
1934, four groups (Budenandt; Slotta; Allen; Hartman) had isolated progesterone and 
determined its structure. 

Dempsey, in 1937, found that injection of progesterone inhibits ovulation in the 
guinea pig. That same year. Makepeace (1937) showed that progesterone inhibits 
ovulation in rabbits, and Kurzrok (1937) and Rock (1937) separately reported the ef- 
fects of these hormones on ovulation in -women. Although the estrogens -were biologi- 
cally active when taken orally, progesterone was shown to have very low activity by this 
method of administration. For this reason, in studies on the antiovulatory effect of 
progesterone, the hormone was administered by injection. Since progesterone is only 
slightly active when taiken orally, an intensive effort was initiated in the mid- 1930 's to 
identify an orally active progestin. 

Attention was turned to dehydroepiandrosterone (DHA), a weakly androgenic ste- 
roid isolated from urine by Butenandt in 1934. In 1935, a number of laboratories 
(Schoeller; Butenandt; Ruzicka; Wallis) reported the preparation of DHA from readily 
available cholesterol. That same year, Oppenauer (1935) prepared DHA from the plant 
sterol sitosterol. This set the stage for the discovery of ethisterone and ethynylestra- 
diol. 

In 193 8, Inhoffen converted DHA to ethynyldiol, which he further converted into 
the 3-oxo-A derivative to which the name ethisterone was given. Inhoffen, that same 
year, condensed acetylene with estrone to obtain ethynylestradiol, a- potent orally active 
estrogen which still remains unchallenged after 30 years of clinical use. Because of 
the androgenic activity of ethisterone, by 1941 it was used only for the treatment of 
severe menstrual disorders (Goldzieher and Rudel, 1974). 

While the search for orally active progestins continued, Russell Marker (1943) de- 
veloped a method for preparing large quantities of progesterone. In 1944, he estab- 
lished Syntex, S. A. , a pharmaceutical connpany which would produce and market pro- 
gesterone (Goldzieher and Rudel, 1974). Marker's technology was responsible for 
driving down the price of progesterone from hundreds of dollars per gram to less than 
10 cents per gram. 



D-3 

Also in 1944, Ehrenstein reported the conversion of the heart glycoside stro- 
phanthidin into a 19-norprogesterone, in an overall yield of 0. 07 percent. This product 
v/as later shown to be not 19-norprogesterone but isomeric with this substance at the 
14- and 17-positions. 

Strophanthidin, the rare glycoside used to make 19-norprogesterone, had to 
undergo a number of chemical reactions to remove the aldehyde group at C-19, result- 
ing in a poor yield. This technical problem was solved by Birch (1944-1947), who de- 
veloped a method for the partial reduction of aromatic compounds. 19-Norprogesterone 
•was shown to be a powerful progestational agent by bioassay in rabbits. This discovery 
of the progestational activity of orally administered 19-norprogesterone constituted a 
major breakthrough in the search for an oral contraceptive. In 1949, Birch synthesized 
a number of 19-nor steroids, including 19-nortestosterone. A major improvement in 
the Birch reduction was discovered by Wilds and Nelson (1953), -who used their modified 
method to obtain 19-nortestosterone in yields of 70 to 77 percent. Although Wilds and 
Nelson did not report their improved methods in the open literature until 1953, they in- 
formed Djerassi at Syntex and Colton at Searle of their improvements in late 1950. 
Djerassi, -who had joined Syntex as Associate Director of Chemical Research (Goldzieher 
and Rudel, 1974), restudied the literature on the 19-nor steroids. Djerassi, Rosen- 
kranz, and Miramontes (1951), working in the laboratories of Syntex, applied the Wilds 
and Nelson procedure in modified fornn. to prepare 19-norprogesterone. Their finding 
that 19-norprogesterone was a nnore potent progestin than naturally occurring proges- 
terone spurred them on to the preparation of norethindrone (norethisterone), an ex- 
tremely potent progestational steroid. 

At Searle, Raymond began working in the area of adrenal steroids (Saunders, 
1975) and developed a method for producing commercial quantities of Cortisol. In the 
early 1950's Searle had an increasing segment of its research staff, under Raynnond's 
direction, devoted to the synthesis of novel steroids and determination of their biologi- 
cal properties. Colton joined the Searle Co. to direct their synthesis effort toward 
new 19-nor steroids in 1951 (Colton, 1975). He quickly prepared a number of 19-nor 
steroids including norethynodrel. In 1953, Saunders, also at Searle, evaluated nor- 
ethynodrel for progestational activity and found it to be surprisingly active when admin- 
istered orally (Saunders, 1957). Drill came to Searle as Director of Biological 
Research in 1953 and played a leading role in directing the biological studies, and the 
metabolic and toxicology studies, leading to clinical testing of the oral contraceptives, 
as -well as other classes of steroids and nonsteroids. 

In 195 1, at the request of a philanthropist, Pincus, a reproductive physiologist at 
the Worcester Foundation, began studying the possibility of controlling fertility by 
chemical means (Goldzieher and Rudel, 1974). With Chang ( 1956), they began testing 
available steroids for antiovulatory activity in 1954. On the basis of the early observa- 
tion by Makepeace (1937) that rabbits fail to ovulate -when injected with progesterone, 
Chang compared the effects of several steroids -which he administered topically, orally, 
and by injection. When Pincus expressed a need for orally active progestins, Searle 
selected norethynodrel to be included in screening studies to measure ability to inhibit 
ovulation in the rabbit. These studies were supported by a grant from Searle (Drill, 
1976). Similar tests -were performed at Searle and norethynodrel was found to be highly 
effective orally in rabbits. A sample of norethindrone forwarded to Chang by Syntex 
also was found to be highly active after oral administration. Thus, both Syntex and 
Searle had orally active progestins. 



D-4 

When progesterone was found to be orally effective in high concentrations in the 
rabbit, Pincus invited Rock, a gynecologist, to work with him on the oral contraceptive 
concept (Goldzieher and Rudel, 1974). Both Searle and Pincus approached Rock in 1954 
with a product that they believed could be clinically effective. A small pilot study in 
1956 showed that ovulation could be suppressed in women by orally administered pro- 
gestin. Because of the anticontraception climate in Massachusetts, Rock decided to 
conduct his large-scale evaluation in Puerto Rico. The oral effectiveness of norethyno- 
drel in -women so exceeded the expectations of Pincus, Chang, and Searle that Searle 
fully supported the Puerto Rican trial of their product Enovid (norethynodrel). The 
trials were funded by Searle and the Planned Parenthood Federation. 

Comparable studies were conducted with norethindrone by Parke-Davis, for 
Syntex (Goldzieher and Rudel, 1974). In 1957, both norethynodrel and norethindrone 
became commercially available for treating menstrual disorders. 

In their initial studies, Pincus and Rock employed the progestins to suppress 
ovulation indefinitely. They soon found that women -who do not menstruate exhibit char- 
acteristic physiological and psychological symptoms, and chronically wonder whether 
they are pregnant. In 1954, Pincus suggested that the progestin be withdrawn for a few 
days each nnonth to permit a normal menstrual cycle (Pincus, 1957). This concept 
was employed with considerable success. 



Combination Therapy 



During the Puerto Rican trials, the investigators were av/are from basic biology 
studies that the norethynodrel preparation used contained some estrogen (Saunders, 
1975), although its significance was not identified until the early clinical trials. When 
problems arose concerning breakthrough bleeding, it was found that the most effective 
preparations contained a trace of the methyl ether of ethynylestradiol (Drill, 1966). 
Mestranol was an impurity carried over from the Birch reduction employed during the 
preparation of norethynodrel. This potent estrogen was reducing breakthrough bleeding. 
Thus, a standard amount of mestranol was added to the formulation of Enovid. This 
allo-wed the amount of progestin used in the preparations to be considerably reduced 
virithout loss of effectiveness. 

In 1959, Searle received approval fro.nn the FDA to market Enovid as an oral con- 
traceptive. The approval of Syntex' s analogous drug was delayed until 1962, largely 
because Parke-Davis and Co. refused to enter the oral contraceptive area (Colton, 
1975). Ortho, in 1962, marketed the Syntex product as Ortho-Novum. Since the intro- 
duction of norethynodrel and norethindrone, approximately 30 varieties of oral con- 
traceptives manufactured by 6 companies have been marketed in the U. S. (Huff and 
Hernandez, 1974), At present, the combination therapy appears to be the most effective 
type of oral contraception. 



Sequential and Minipill Therapy 



T-wo other types of oral contraceptive therapy have also been introduced — sequen- 
tial and minipill. Sequential therapy involves the adnn.inistration of estrogen alone for 
the first 15 days, with progestin being added during the last 5 or 6 days. It was 



D-5 

introduced in 1963 by Mead-Johnson (Huff and Hernandez, 1974) and employs dimeth- 
isterone, synthesized and evaluated by the British Drug Houses, Ltd. , in 1954-1957 
(Petrow, 1970), as the progestin. Goldzieher and Rice-Wray established that 
ethynylestradiol possessed unique advantages as an ovulation inhibitor in studies in 
Mexico in 1960. Four types of sequential formulations are now in use - tvifo are manu- 
factured by Mead- Johnson, and employ ethynylestradiol as estrogen, and dimethi- 
sterone as progestogen, while the other two employ mestranol and norethindrone and 
are manufactured by Ortho and Syntex. 

In 1972, the FDA approved the first progestin-only oral contraceptive, the mini- 
pill (Huff and Hernandez, 1974). Unlike the others, this type is administered on a 
continuous daily basis. Two varieties, using 0.35 mg norethindrone, are manufactured 
by Ortho and Syntex. They are the least effective of all the oral contraceptives novv^ 
in use. 



Side Effects 



A number of potential side effects of the use of oral contraceptives have been 
studied. Much of the published infornaation is still conflicting and inconclusive. How- 
ever, as early as 1961, Jordan reported isolated cases of thromboembolism among 
oral-contraceptive users. A 4-year study by Sartwell ( 1969) indicated that the risk of 
thromboembolism is increased. It is connparable to that occurring during pregnancy 
and is slightly greater for sequential-therapy users than for combined. There is no 
evidence for carcinogenicity (Andrews, 1971). It has been suggested that oral- 
contraceptive agents may instead exert a protective influence on the endometrium and 
possibly the breast, two tissues once thought to be adversely affected by the oral 
agents. 

Glucose tolerance is lowered in the first fe-w months of use (Spellacy, 1969); 
these changes are clinically significant only in v^romen with latent diabetes and are re- 
versible with discontinued use of oral contraceptives. Hypertension may be induced 
in an occasional susceptible individual, but it, too, is reversible (Andrev/s, 1971). 

There is an observed dose-related increase in sulphobromophthalein retention 
(BSP) in women -with inherited or acquired defects in hepatic excretory function 
(Mueller and Kappas, 1964; Eisalo et al, 1965; Gallagher et al, 1965; Ockner and 
Davidson, 1967; Roman and Hecker, 1968). Ophthalmologic al abnormalities are no 
more frequent in users than in nonusers (Andrews, 1971). Thyroid function is not 
changed, although some tests are influenced by oral contraceptives. Migrain headaches 
may be initiated or aggravated (Nicholson and Walsh, 1969). These and other possible 
etiological effects are difficult to assess. 



D-6 

REFERENCES 

Anon., FDA Drug Bull., 3 (December, 1972). 

Albright, F. , in Internal Medicine — Its Theory and Practice , E. Musser(Ed. ), Lea 
and Febiger, Philadelphia, Pa. (1945). 

Allen, E. , and Doisy, E. A., J. Amer. Med. Assoc, 8J_, 819 (1923). 

Allen, W. M. , and Wintersteiner, O. , Science, 80, 190(1934). 

Andrews, W. C. , Obstet. Gynecol. Surv. , 2i6, 477(1971). 

Birch, A. J., and Robinson, R. , J, Chem. Soc., 403 (1944). 

Birch, A. J., J. Chem. Soc, 809 (1945). 

Birch, A. J., J. Chem. Soc, 593 (1946). 

Birch, A. J., J. Chem. Soc., 102(1947). 

Birch, A. J., J. Chem. Soc., 1642(1947). 

Birch, A. J., and Mukherji, S. M. , J. Chem. Soc, 2531 (1949). 

Butenandt, A., Ber. , 63, 659(1930). 

Butenandt, A., Ber., 64, 2529(193 1). 

Butenandt, A., Dannenbaum, H. , Hanische, G. , and Kudszue, H. , Z. Physiol. Chem., 
237 , 57 (1935). 

Butenandt, A., and von Ziegner, E. , Z. Physiol. Chem., 188, 1 (1930). 

Butenandt, A., Westphal, U. , and Hohlweg, W. , Z. Physiol. Chem., 227, 84(1934). 

Colton, F. , Searle Laboratories, Chicago, Illinois, Personal Communication, 
December 11 (1975). 

Corner, G. W. , and Allen, W. M. , Amer. J. Physiol., 88, 326 (1929). 

Dempsey, E. W. , Amer. J. Physiol., 120 , 126(1937). 

Djerassi, C. , Rosenkranz, G. , Iriarte, J., Berlin, J., and Romo, J., J. Amer. 
Chem. Soc, 73_, 1523 (1951). 

Djerassi, C. , Rosenkranz, G. , and Miramontes, L. E. , U. S. Patent 2, 759, 951 (1956) 
[claims priority June 4, 1951 (Mexico)], Application October 5, 1951. 

Doisy, E. A., Veler, C. D. , and Thayer, S. A., Amer. J. Physiol., 90, 329 (1929). 

Doisy, E. A., Veler, C. D. , and Thayer, S. A., J. Biol. Chem., 86, 499(1930). 



D-7 

Drill, V. A. , Oral Contraceptives , McGraw-Hill Book Co. , New York ( 1966). 

Drill, V. A., M.D., Personal Communication, January 7 (1976). 

Eisalo, A., Jarvinen, P. A., and Luukkainen, T. , Brit. Med. J., J_, 1416 (1965). 

Ehrenstein, M. , J. Organic Chem. , 9, 435(1944). 

Fellner, O. O. , Arch. Ges. Physiol. (Pflugers), 189 , 199 (192 1). 

Fellner, O. O. , Biochem. Z., 147 , 184(1924). 

Fellner, O. O. , German Patent 486, 9 14, May 15 (1924). 

Fellner, O. O. , Deut. Med. Wochschr. , 54, 922(1928). 

Gallagher, T. F. , Mueller, M. N. , andKappes, A,, Trans. Assoc. Amer. Physicians, 
7_8, 187 (1965). 

George Washington University Medical Center, Population Report, Series A, 1, A-1 
(1974). "~ 

Goldzieher, J. W. , and Rudel, H. W. , J. Amer. Med. Soc. , 230 , 42 1 (1974). 

Haberlandt, L. , Munch Med. Wochschr., 68, 1577(1921). 

Haberlandt, L. , Arch. Ges. Physiol. (Pflugers), 202 , 1(1924). 

Haberlandt, L. , Arch. Ges. Physiol. (Pflugers), 2^6, 525 (1927). 

Hartmann, M. , and Wettstein, A., Helv. Chim. Acta, JJ, 1365(1934). 

Huff, J. E., and Hernandez, L; , J. Amer. Pharm. Assoc, l^SM, 244(1974). 

Inhoffen, H. H. , Logemann, W. , Hohlweg, W. , andSerini, A., Ber. , 7J_, 1024(1938). 

Jordan, W. M. , Lancet, 2, 1146 (1961). 

Kurzrok, R. , J. Contraception, 2, 27 (1937). 

Makepeace, A. W. , Weinstein, C. L. , and Friedman, M. H. , Amer. J. Physiol., 
119 , 512 (1937). 

Marker, R. , U. S. Patent 2, 29 1, 643, August 4 (1 943 ). 

Marrian, G. F. , Biochem. J., 23_, 1090(1929). 

Marrian, G. F. , Biochem. J., 24, 435(1930). 

Mueller, M. N. , and Kappas, A., J. Clin. Invest., 43, 1905(1964). 

Nicholson, D. H. , and Walsh, F. B. , J. Reprod. Med., 3_, 37 (1969). 

Ockner, R. K. , and Davidson, C. S. , Brit. Med. J., 276,33 1 (1967). 



D-8 

Ockner, R. K. , and Davidson, C. S. , N. Engl. J. Med., 276 , 331 (1967). 

Oppenauer, R. V., Nature, 135 , 1039 (1935). 

Petrow, v.. Essays in Biochemistry, 2, 117 (1966). 

Petrow, v., Chem. Rev., 70, 713 (1970). 

Pincus, G. (Ed. ), Recent Progress in Hormone Research, Academic Press, Inc. , 
New York (1954). 

Pincus, G. , Proc. Symp. 19-Nor-Progestational Steroids, Searle Research Labs., 
Chicago, 105 (1957). 

Pincus, G. (Ed. ), Recent Progress in Hormone Research , Academic Press Inc. , 
New York, 323 (1957). 

Pincus, G. , Chang, M. C. , Hafez, E. S. E. , Zarrow, M. X., and Merrill, A., 
Science, 124 , 890 (Nov. , 1956). 

Rock, J., and Barlett, M. K. , J. Amer. Med. Assoc, j^, 2022 (1937). 

Rock, J., Pincus, G. , and Garcia, C. R. , Science, 124, 891 (November, 1956). 

Roman, W. , and Hecker, R. , Med. J. Aust. , 2, 682(1968). 

Ruzicka, L. , and Wettstein, A., Helv. Chim. Acta, ^l, 986(1935). 

Sartwell, P. E. , Masi, A. T. , Arthes, F. G. , Greene, G. R. , and Smith, H. E. , 
Amer. J. Epidem. , 90, 365(1969). 

Saunders, F. J., Endocrinology, 60, 804(1957). 

Saunders, F. J. , Director, Research Liaison, Searle Laboratories, Chicago, Illinois, 
Personal Communication, December 11 (1975). 

Schoeller, W. , Serini, A., and Gehrke, M. , Naturwissenschaften, 23, 337 (1935). 

Slotta, K. H. , Ruschig, H. , and Fels, E., Ber. , 67, 1270(1934). 

Spellacy, W. N. , Amer. J. Obstet. Gynec. , 104 , 448(1969). 

Wallis, E. S., and Femholz, E. , J. Amer. Chem. Soc. , 57, 1379 (1935). 

Wilds, A. L,, and Nelson, N. A., J. Amer. Chem. Soc, 7_5, 5360, 5366(1953). 

Wintersteiner, O. , and Allen, W. M. , J. Biol. Chem., 107, 321(1934). 



\ \\tiS\Z 



;1':c|j,-jnl 



: riK CI; is! 'lOi bo 






o\q\u\f 



vile .:. n-. 



1954\ British Drug Houses, Ud. 
1957 j Synthesis of dimethisterone 
NR/S/W/P/C 

1957 British Drug Houses, Ud, 

Evaluated gynecologicd! proper- 
ties ot dimethisterone 

MR/D/W/P/C 



ESTROGENS 

- 1921 ( Feilner Showed that extracts from 
1927^ ouerian tissues had etio- 

logical effects 

NR/s/w/ m 



- 1923 Ooisy Developed estrogen 
bio assay 

NR/SAIV/ / 



- 1929 Doisv isolated estrogen from 
NR/S/U/ / 

-1930 Doisy Determined the structure 

Butenandt of estrone and estriol 
Marrian 

NR/D/ / / 

-1934 Butenandt Isolated DHA from 

NR/S/W/ / 

"1935 Schoeller Prepared DHA from 
Butenandt cholesterol 
Ru7ick3 
Wallis NR/D/W/P/M 



PROGESTINS 



•aluation and 



1 



195?) Goldzieher 
1961 ( Maas 

Maitinez-Manautou mestranol alone as 
Rice-Wray oral contraceptive 

Rudel with progestin as 

Spain menstrual regulator 

(sequential) 
MR/D/U,W/P/iVl,l 



1963 Mead Johnson Introduction of sequential 
oral contraceptive therapy 
DV/D/W/P/C 



-1938 Inhoffen Converted DHA to ethisterone; 
also synthesized ethynylestradiol 
NR/D/W/ /C 



- 1945 Albright Oral preparations of estrogen 
and progesterone attempted 
for endocrinal disorders 
NR/D/ / / 



-1921 ( Haberlandt Induced si 
1927/ ovarian e=. 



-1957 ( ( 
1960 f I 



1969 Sartwell Incidence of thromboembo- 
lism is greater for sequential 
therapy users than for com- 
bined therapy 

MR/S/ / / 



. Goldzieher Evaluation of mestranof - 
Rice-Wray and ethynylestradiol as 
an inhibitor o( ovulation 
MR/D/U.W/ /I 

- 1957 FDA Approval and marketing of 

Searle progestin /estrogen (combina- 
tion) EfyOVIO, for menstrual 
disorders 

DV/S/U/G,P/GL,C 
-1959 FDA Approval of ENOVIO for oral 
contraception 

DV/D/U/G/GL 

- I960 Searle Introduced ENOVID for use as 

an oral contraceptive (combina- 
tion therapy) 

DV/D/U/P/C 

- 1961 Jordan Isolated case reports of thrombo- 

embolism were reported among 

NR/S/U/P/M 

- 1964 Mueller Observed a dose-related 
1965 Eisalo increased BSP retention in 
1965 Gallagher oral contraceptive users 

1967 Ockner 

1968 Roman MR/S/U.W/G,P/M 



NR/S/W/ /M 



-1929 Corner Developed bioassay for 
progesterone 

NR/S/U/P/C 



— 1934 Butenandt Isolated and determined 

Slotta the structure of progesterone 

Allen 

Hartmann NR/D/ / / 

— 1937 Makepeace Progesterone inhibits 

Oempsey ovulation in guinea pigs 
NR/D/ / / 

— 1937 Rock Progesterone inhibits 

ovulation in women 

NR/D/U/ m 

—1943 (Vlarker Method for large-scale 

synthesis of progesterone 
NR/D/W/P/C 

— 1944 Marker Established Syniex, 



S.A, 



MR/D/W/P/C 



-1950 Colton (Searle) Synthesized 
norethynodrel 

MR/D/U/P/C 
- 1951 Pincus Tested norethynodrel and 
Chang norethindrone for progestin 
activity when given orally 
to animals 

MR/D/U/P/I,C 
Rock Tested norethynodrel and 
norethindrone in women 
MR/D/U/P/C,M 



-1952 ( 
1957 j 



-19651 Sartwell Rclati 
1969/ 



of thromtwembo- 
lism for oral contraceptive users 
is 4,4 times that of nonusers 
MR/S/U/G/M 



—1969 Spellacy Glucose tolerance is initially 
diminished in women using 
oral contraceptives, it returns 
to normal with long-term use 
MR/S/U/ m 

—1969 Nicholson Migraine can be precipi- 
tated or aggravated by use 
of oral contraceptive 
MR/S/U/ /M 



- 1972 FDA Approval of the usD of progestin 
only oral contraceptive agent 
(minipill) 

DV/D/U/G/GL 



- 1944 Ehrenstein Prepared 19-norprogestero 

showed It to be a strong 
progestin 

NR/S/U/P/M 

- 1949 Birch Improved method for making 

19-nor steroids 

NR/S/W/ / 

- 1950 Wilds Improved BIRCH reduction 

method 

NR/S/U/P/M 
-1951 Djerassi (Syntex) Synthesized 
norethindrone 

MR/D/W/P/C 



CODES 
NR Nonmission-Oriented Research 

MR Mission-Oriented Research 

DV Development 

S Significant Event 

D Decisive Event 

U U.S. Event 

W Foreign Event 

G Government Funding 

P Private Funding 

M Medical School - Hospital ~ 

University 

I Research Institution 

GL Government Laboratory 

C Company 



HISTORIOGRAPH-ORAL CONTRACEPTIVES 



nspoina baqolavsQ vz'oC 



-1 



E-1 

APPENDIX E. PENICILLIN 

INTRODUCTION AND SUMMARY 



The development of penicillin and the introduction of penicillin therapy into clini- 
cal medicine completely revolutionized the treatment of bacterial infections in man. 
Not only was penicillin itself a remarkably povi^erful therapeutic v/eapon, but its suc- 
cessful development laid the groundvi^ork for the discovery and development of other 
"miracle drugs " which followed: streptomycin, chloroamphenicol, aureomycin, and 
terramycin. Out of this one development grew a multibillion dollar industry and, more 
importantly, a mode of therapy -which has saved millions of lives. 

The original discovery of penicillin occurred in the modest, ill-equipped labora- 
tory of Alexander Fleming at St. Mary's Hospital, London, in 1928. It remained little 
more than a laboratory curiosity for another decade. While Fleming recognized that 
this material -which -was produced by a contaminating moid might have potential as a 
medicinal, he -was discouraged by the minute yields of material and its extreme insta- 
bility. Realizing that he did not have access to the staff and facilities that would be re- 
quired to advance the development, and following publication of the limited amount of 
-work he was able to accomplish, he became resigned to using the material only as a 
laboratory aid in his work on isolation of specific microorganisms. 

Professor Florey at Oxford had access to an interdisciplinary team and -when his 
laboratory became interested in Fleming's mold broth in 1938, they quickly made a 
quantum jump in the development. Nevertheless, imnnense problems in biology and en- 
gineering remained to be solved before penicillin -would become a practical reality. 
World War II forced Florey to bring this program to the United States for assistance in 
1941. This assistance was found in the Northern Regional Research Laboratory of the 
U. S. Department of Agriculture. 

What folio-wed -was an unprecedented effort involving hundreds of scientists and en- 
gineers in universities, industry, and government laboratories which culminated in the 
successful comnaercial production and -widespread distribution of penicillin by 1945. 



HISTORICAL ACCOUNT 



The story of the development of penicillin as an antibiotic is one of an unexpected, 
spectacular discovery in 1928 foUo-wed by 10 years of relative disinterest and inactivity, 
folio-wed in turn by a virtual explosion of -work -which resulted in -widespread clinical ap- 
plication by 1945. To appreciate the full significance of the discovery and amazing de- 
velopment of penicillin, it is important to examine the events -which preceded its dis- 
covery as -well as the -work -which led to its clinical acceptance and large-scale 
production. 



E-2 

Background 



The existence of naturally occurring antimicrobial substances was not unknown 
prior to Alexander Fleming's discovery of penicillin in 1928. As early as 1877, 
Pasteur had reported that the growth of anthrax bacteria was arrested by the presence 
of contaminating organisms in the culture broth. He subsequently dennonstrated that 
rabbits that -were simultaneously injected with anthrax and an antagonistic E. coli strain 
did not contract the disease. In 1899, Emmerich and Lb'w reported that a substance 
produced by Bacillus pyocryaneus (Pseudomonas aeruginosa) was detrimental to other micro- 
organisms. At the Pasteur Institute, Metchnikoff recognized the significance of this 
finding and encouraged further work on the material. It -was not until 192 1, however, 
that Goris and Liot succeeded in isolating and demonstrating the properties of pyocya- 
nin, one of the active constituents of the mixed chemical substance produced by this 
organism. Two other chemicals vi^ere subsequently isolated, one that had some effec- 
tiveness against diphtheria and another -which destroyed the cholera germ. In spite of 
these discoveries, the general concept of fighting one germ with another was foreign to 
the medical community and the idea v/as generally suppressed. 

Prior to the outbreak of World War 1, the pioneering vifork of Pasteur and 
Dr. Edward Jenner on the use of vaccines was gaining rapid acceptance and the medi- 
cal profession had adopted Joseph Lister's ideas on the use of antiseptics in the op- 
erating room. Indeed, the use of aseptic techniques in surgery had replaced the use of 
chemical antiseptics, except for the occasional treatment of septic -wounds -which -were 
still treated with dressings of carbolic acid, mercury salts, and boric acid. During 
the war years, surgeons for the first time were faced with treating thousands of septic 
wounds which did not respond to treatment by topical antiseptics; nevertheless, each 
antiseptic continued to have strong groups of supporters among the medical community. 

Prior to World War I, Alexander Fleming had begun -working as a bacteriologist 
at St. Mary's Hospital, London, under the direction of Almroth Wright -who -was a strong 
disciple of Pasteur and a champion of the future of immunization for disease control. 
Fleming was strongly influenced by Wright's belief that the most potent defense against 
disease-causing microorganisms was the body's own protective mechanism. When the 
war began, Fleming followed Wright to Boulogne where he became deeply involved in 
the study of septic wounds and the use of antiseptics. As a result, he became a strong 
antagonist of antiseptics based on his observation that they -were often more harnnful to 
the body's protective mechanisms than to the nnicroorganisms they were meant to de- 
stroy. Following the war, Fleming continued to study the body's natural defense sys- 
tem. In 1922, he discovered lysozyme, the antimicrobial material in human tears, 
saliva, and sputum (Fleming, 1922). This discovery kept alive his interest in natural 
nnaterials having antimicrobial properties. 

Meanwhile, Gratia and Dath (1925), working with Staphylococcus aureus at the 
Pasteur Institute, had discovered a fungus which killed Staphylococcus. They also noted 
that they had observed a Penicillium mold which had a similar effect. However, they did 
not isolate the latter mold and their finding attracted little attention. Then, in 1928, 
Fleming observed the same phenomenon on a culture plate with Staphylococcus aureus. 
Because of his previous experience with lysozyme, he was sufficiently intrigued by the 
bacteria-lysing properties of a contaminating mold on the culture plate that he isolated 
it and began to investigate its antagonistic properties. 



E-3 

Early Work 



Fleming quickly established that the mold produced a substance which -was antag- 
onistic to many Gram-positive organisms while having a remarkably low toxicity in ani- 
mals. He was particularly impressed with the material since it did not harm leukocytes 
as did the common chemical antiseptics. The mold v^as incorrectly identified as 
Penicillium rubrum and he named the active substance penicillin. 

Fleming published his discovery in 1929 in the British Journal of Experimental 
Pathology. He then organized a small team to study the substance in greater detail. 
They had little success and Fleming became discouraged because he recognized that the 
material -was extremely labile and that it -would never be of practical use unless it could 
be isolated in a stable form. He v^as -working in a microbiology laboratory and there 
-were no chemists on the staff to assist in this problem. He had little equipment and no 
funds for the work. While he continued to talk to others about the striking properties of 
penicillin and its potential value as a chemotherapeutic agent, he himself discontinued 
his efforts to isolate and identify the active principle. In his laboratory penicillin con- 
tinued to be used only as a tool in his work on the selective isolation of bacteria { 1932). 
While there has been much speculation and many theories offered on -why Fleming did 
not pursue this development more vigorously, the single most important deterrent 
seems to have been the extreme instability of the material itself (Hare, 1970), which he 
was not able to overcome. 

Fleming's choice of a pathology journal for the publication of his discovery -was 
perhaps unfortunate, for it -was not widely read by mycologists and chemists who might 
have joined Fleming in the study of penicillin. Professor Harold Raistrick at the London 
School of Hygiene and Tropical Medicine was one of the fe-w chemists -who learned of 
Fleming's discovery and -was sufficiently interested to assemble a small team to isolate 
the material. Under Professor Raistrick's direction, this group developed a synthetic 
medium for growing the mold and developed the first crude procedure for extracting 
the penicillin (Clutterbuck, et al. , 1932). They reported several important findings 
on the instability of penicillin in acid and alkali solutions. They also submitted the 
mold to Dr. Charles Thom at the U. S. Department of Agriculture who correctly identi- 
fied it as Penicillium notatum Westling. This -was a key development, for in later years it 
-would properly set the stage for the search for more productive strains of the mold. It 
is not clear -why Raistrick discontinued -work on penicillin at this point. Part of the rea- 
son -was undoubtedly that the mycologist on the team -was killed in an accident and the 
bacteriologist accepted another appointment. Physicians -whom he approached \vere not 
interested in testing this ne-w material (Raper, 1975) and Raistrick eventually -went on 
to other things. 

The only other interest recorded -was by Roger Reid at the Pennsylvania State Col- 
lege who laboriously examined dozens of molds and demonstrated the uniqueness of some 
molds of the genus Penicillium in producing the inhibitory substance (1933). Reid (1935) 
-was also the first to sho-w that penicillin -was bacteriostatic rather than bactericidal and 
he proceeded to characterize several key properties of the substance. Nothing further 
-was done and penicillin -was all but forgotten. 



E-4 
Renewed Interest 



Seven years after Fleming's report, Weindling and Emerson (1936) in California 
isolated an antifungal material produced by Trichoderma lignorum, and in 1939 (a, b, c), 
Dubos, at the Rockefeller Institute for Medical Research, discovered a bactericidal 
agent (tyrothricin) produced by a soil bacillus (B. brevis) which was effective against pneu- 
mococci. Although tyrothricin was shown to be insoluble in body fluids and toxic to body 
tissues, its discovery by Dubos received considerable attention and was responsible in 
part for renevi^ed interest in Fleming's discovery. 

During the period 1922 to 1938, Professor Howard W. Florey and his colleagues at 
the Sir William Dunn School of Pathology, Oxford, had been researching the chemical 
action of Fleming's lysozyme. With continued interest in substances which lysed micro- 
organisms, Professor Florey and Dr. Ernest B. Chain, in 1938, began looking for other 
sources of such materials. Searching the literature, they came upon Fleming's 1929 
paper and selected penicillin as the first material to study. They had not been aware of 
this material before this tinne. By 1940, they had published their initial results (Chain, 
et al. , 1940) on the chemotherapeutic properties of penicillin in animals, and in 1941, 
they published their classical paper (Abraham, et al. , 1941) which described the cul- 
tural conditions and extraction procedures for small-scale production of penicillin; data 
on the absorption, excretion, and low toxicity in animals; the cylinder assay method; 
its antibacterial properties; and the first human clinical trials. This was certainly the 
most decisive event in the development of penicillin since Fleming's original discovery. 
In essence, penicillin experienced a rebirth. 

World War II had arrived in England almost simultaneously with the rediscovery of 
penicillin. The war greatly magnified the potential value of producing this new antibiotic 
material in large quantities. At the same time, it created a situation in England which 
all but precluded the allocation of resources and manpower -which -would be required for 
the development. The -work of Florey and his colleagues had identified the basic prob- 
lems that -would need to be resolved before penicillin could be considered a practical 
medical tool. Under the culture conditions employed, the active material was produced 
in very small amounts. Using surface culture techniques, it appeared that thousands of 
containers of culture would be needed to produce sufficient material even for meaningful 
clinical trials. Because of the war-induced shortage of the needed materials and the 
nnanpo-wer -which -would be required for such a laborious production procedure, this -was 
a very serious deterrent to the continued development of penicillin. 

The studies of Florey and his co--workers had been supported in part by funds from 
the Rockefeller Foundation. In 1941, Professor Florey and Dr. Norman Heatley came to 
the United States on a Rockefeller Foundation grant to solicit assistance in continuing the 
developnnent of penicillin. This decision proved to be decisive to the outcome of the 
progrann. 

In the United States, Florey and Heatley were directed to contact the National 
Academy of Sciences in Washington where they -were referred to Dr. Charles Thom. 
Dr. Thonn. then arranged for them to go to the Northern Regional Research Laboratory 
(NRRL) of the U. S. Department of Agriculture which had a newly organized Fermenta- 
tion Division. There they quickly obtained the support of Dr. O. E. May, Director 
of the Laboratory, and Dr. R. D. Coghill, head of the Fermentation Division. Work 
was initiated on three fronts. First, the nutrition of the mold in surface cultures 



E-5 

was studied to find ways to improve the yield of penicillin. Second, studies were begun 
on the feasibility of producing the material in submerged culture. Third, a search for 
other, more productive strains was initiated. 

At this point it becomes impossible to construct a meaningful and accurate flow of 
events in absolute chronological sequence. Many things began happening at once. While 
the NRRL was simultaneously pursuing the three lines of development cited above, 
Florey visited several pharmaceutical companies to solicit their help in producing suf- 
ficient material for clinical trials. The U. S. Government became involved through the 
Committee on Medical Research (CMR) of the Office of Scientific Research and Develop- 
ment (OSRD). By 1943, the responsibility for the production of penicillin by fermenta- 
tion methods was assumed by the War Production Board while the CMR retained control 
over the programs on synthesis of penicillin. The Committee on Therapeutic and Other 
Agents of the National Research Council coordinated the distribution of all penicillin for 
clinical testing. 

Early in 1942, sufficient penicillin had become available to treat 10 clinical cases. 
By June, 1944, there v/as a sufficient supply to treat all of the casualties of the D-Day 
invasion, and by March, 1945, distribution was begun through civilian commercial chan- 
nels (Richards, 1964). This fantastic accomplishment in fermentation technology was 
made possible through the combined research and development efforts of over 20 com- 
panies, 5 universities, and numerous government agencies in the United States (Elder, 
1970). These efforts were also shared with the British governnnent which, in turn, 
supplied reports on the continuing, albeit less intense, efforts being carried out in that 
country. 



Key Developments 



For purposes of this report, the key developments that led to the large-scale 
clinical distribution of penicillin have been grouped into three broad categories: 

(1) studies on the toxicity and chemotherapeutic properties of penicillin in animals; 

(2) advances in fermentation technology, including production methods, isolation of im- 
proved mold strains, and innproved assay methods; and (3) human clinical trials. Since 
the work at the NRRL has only been published in review articles which cover multiyear 
research efforts, the publication dates do not place the developments in proper histor- 
ical sequence and the historiograph citations are marked with the time frame of the 
actual work in addition to the year of publication. 



Animal Studies 

The material used by Abraham, et al. , in 1941 for the initial therapeutic trials 
contained 40 to 50 Oxford units/mg of penicillin. By 1942, material containing 500 
Oxford units/mg was available and Florey and Jennings (1942) reported that the toxicity 
in aninnals -was reduced even further v/ith this purer, more potent material. In the 
U. S. , Hobby and co-workers at Pfizer ( 1942a) confirmed and extended the work of the 
Oxford group on the antimicrobial powers of penicillin in vitro and demonstrated its 
chemotherapeutic properties in mice (1942b). They also administered sodium and am- 
monium salts of penicillin to humans with no untoward effects. At the Merck Institute 
for Therapeutic Research, H. J. Robinson (1943) reported the toxic dose of crude peni- 
cillin to be about 64 times the effective dose as determined by subcutaneous injection 
in mice. 



E-6 

Fermentation Technology 

During this same period of time, critical advances were being made at the NRRL 
on the production of penicillin. One. of the most significant developments early in the 
program was the demonstration by Moyer that the yield of penicillin in surface cultures 
could be increased severalfold by the use of corn steep liquor in the media, a material 
that had been previously used successfully as a substitute for yeast extract in the sor- 
bose fermentation (Wells, 1939). Under the direction of Moyer and Coghill (1946a), the 
yield of penicillin was soon increased 100-fold by combining the use of corn steep liquor 
with lactose as the principal carbohydrate and by the addition of other nutrients during 
the course of the fermentation. 

While these advances in culturing techniques were being naade, a second group 
under the direction of Dr. Kenneth Raper at NRRL was isolating and characterizing 
high-yielding mold cultures. Beginning with the Fleming strain, Raper and his co- 
workers isolated dozens of substrains. The most productive was NRRL 1249. B21. 
They then turned their attention tow^ard the isolation of new strains from moldy foods 
and soil samples collected around the world (Raper, et al. , 1944; Raper and Alexander, 
1945). The U. S. Army as well as many private citizens contributed to this collection 
process. One highly productive strain (NRRL 1941) was isolated from a moldy canta- 
loupe. This strain became the parent of several substrains and mutants which were de- 
veloped at NRRL and at other laboratories under the sponsorship of the Office of Pro- 
duction Research and Development. Mutant X-1612, produced at Carnegie Institute, 
tested at the University of Minnesota, and fully established by pilot plant work at the 
University of Wisconsin, was an outstanding example of the value of these collaborative 
efforts (Raper, 1947). This X-ray-induced mutant increased production to 500 /i/ml 
of penicillin. The yield was further doubled by the mutant produced in 1945 by ultra- 
violet irradiation at the University of Wisconsin, Q-176 (Backus and Stauffer, 1955). 

As each new strain or mutant was discovered which substantially increased 
yields, either in surface culture or submerged culture, it ■was made available to those 
industrial companies that were struggling to produce sufficient product first for clinical 
trials and then for wartime needs. Without these key developments in mycology, 
large-scale production would have been impractical if not impossible. 



Submerged Culture Development . The production of penicillin by surface culture 
techniques, even with the advances in culture media and highly productive strains, re- 
quired thousands and thousands of small containers which had to be individually pro- 
cessed by hand. While entire production facilities based on this technique were estab- 
lished in an effort to meet quickly the demand for penicillin, it was obvious that it 
would be highly preferable to use submerged culture techniques in large-scale fer- 
menters. At NRRL, Moyer and Coghill (1946b) first demonstrated the feasibility of 
producing penicillin in submerged culture using a special strain of P. notatum Westling 
(NRRL 832). 

The work of NRRL, beginning in 1942, was communicated monthly to the CMR of 
the OSRD. From here, the information was distributed to other research groups and 
the industrial producers. The problems v^hich had to be solved fronm the initial dem- 
onstration of feasibility to actual commercial application were formidable. Never be- 
fore had deep tank fermentations been performed which required the introduction of 
vast quantities of sterile air, and presented the associated problems of mixing and buf- 
fering. Critical developments were required in harvesting techniques, extraction 



E-7 

processes, recovery of solvents, and drying of the final product. Hundreds of engi- 
neers and scientists in many organizations were working simultaneously to solve the 
many problems. They continued to share each advance with all concerned. Because 
of the vastness of the effort, it is not possible to pinpoint specific key developments in 
this area. Elder (1970) has reported a broad historical account of some of these 
activities. 



Assay Methods 

As advances were made in culturing techniques, mold strains and fermentation 
technology, it was important that an assay method be available for measuring the 
changes in yield. Fleming's technique for measuring the potency of his mold broths 
was largely qualitative. The first quantitative assay was developed by Heatley in 
Florey's laboratory (Abraham, et al. , 1941). This was the cylinder-plate test which 
was so extremely useful during the initial stages of penicillin development. It was used 
to express the potency of penicillin preparations in terms of the Oxford or Florey unit 
which was defined in terms of the amount of penicillin required to inhibit the growth of 
staphylococcus in a zone 24 mm in diameter under specified conditions. This basic 
method was subsequently modified by Foster and Woodruff ( 1943) and Schmidt and 
Moyer (1943) to improve its accuracy and reproducibility. About the same time, tur- 
bidometric procedures were refined by Lee, et al. (1944), at Wallace Laboratories, 
Inc., Joslyn (1944) at Parke Davis and Company, and McMahan (1944) at Chas. Pfizer 
and Company, Inc. In October, 1944, at a conference held by the Health Organization 
of the League of Nations, the international unit of penicillin was defined as the activity 
contained in 0. 6 microgram of an international standard of pure sodium penicillin G, 
which had been prepared by Dr. Stadola, a chemist at NRRL. 



Clinical Studies 

In his original paper, Fleming (1929) referred to the lack of toxic symptoms fol- 
lowing the topical treatment of infected surfaces in man and the lack of irritation fol- 
lowing irrigation of the human coQJunctiva with his mold broth. He gives no further 
indication of the therapeutic values of these treatments. Thus, it was not until the re- 
port in 1941 on the treatment of 10 patients by the Oxford group that the chemothera- 
peutic properties of penicillin were demonstrated in humans (Abraham, et al. , 1941). 
In spite of the very small amounts of very impure material available to these -workers, 
the results were sufficiently dramatic to convince Florey and his colleagues that the 
development of penicillin must continue. 

Even as Florey and Heatley traveled to the U. S. , the Oxford team continued to 
produce penicillin by surface culture. With assistance of Imperial Chennical Industries, 
they produced sufficient material to treat 187 patients during the next year and a half 
(Florey and Florey, 1943). The success of these trials led to the formation of the Gen- 
eral Penicillin Committee in Britain for the control of penicillin production. 

In the U. S. , clinical trials proceeded slowly at first and then increased dra- 
matically as nnaterial became available. Dr. Chester S. Keefer, as Chairman of the 
Committee on Therapeutic and Other Agents of the National Research Council, as- 
sumed the responsibility for coordinating all civilian tests using the materials supplied 
by the CMR of the OSRD. The CMR began soliciting the involvement of commercial 
firms in October, 1941, just 3 months after Florey's arrival in the U. S. (Richards, 



E-8 

1964). Merck and Co. , Inc. , expressed immediate interest and began work. They 
agreed to share whatever information they could within the constraints of the Sherman 
Antitrust Laws. Because of this legal constraint and the very low yields which were 
possible by existing technologies at that time, other companies were reluctant to 
make a definite commitment. However, by February, 1942, E. R. Squibb and Sons 
joined Merck in a formal agreement to undertake a collaborative program of penicillin 
research and development. Chas. Pfizer and Co. joined the group in Septemiber of 
that year, as did many other companies soon thereafter. This industrial involvement 
and cooperation made possible the early availability of penicillin for clinical trials. 

The first patient was treated in the U. S. in March, 194Z, using material sup- 
plied by Merck. By February, 1943, 90 additional cases had been treated and by 
August, 1943, the results of the treatment of 500 civilian cases were published by 
Dr. Keefer. In December of the same year, Rammelkamp and Keefer (1943) reported 
in detail on the absorption, excretion, and distribution of penicillin in humans when 
administered by various routes. 

The treatment of military patients began in April, 1943, and by December, 
Lyons reported on the treatment of 209 patients. The results of this study convinced 
the Army of the value of penicillin treatment. Also in December, 1943, Mahoney et 
al. , reported the initial favorable results of treating four patients at the U. S. Marine 
Hospital for priraary syphilis. 

During the latter half of 1943, the War Production Board began organizing indus- 
try for greatly increased production. Twenty-one companies were now included in the 
effort. Production rose from 400 million units in the first half of 1943 to 20. 5 billion 
units during the latter half. In May, 1944, limited distribution to civilians began and 
by March, 1945, distribution through ordinary commercial channels had begun. 



Synthesis 

In the preceding historical summary, little has been said about the efforts toward 
penicillin synthesis. This is not because of lack of activity in this area. Indeed, a 
massive collaborative effort was conducted in this area during the same time frame as 
the efforts on fermentation technology. While the work was closely guarded during the 
war years, a compendium of approximately 700 reports was compiled and published in 
1949 (Clark). While iTiuch very good work was done in this area, practical synthesis 
was not achieved during the war, and the work only indirectly affected the total develop- 
ment program for the most part. One example of significant spinoff was the addition 
of phenylacetic acid, which had been identified as a degradation product of penicillin, 
to fermentation media. This significantly increased the yields, and enabled such high- 
yielding strains as X-1612 and Q-176 to produce more penicillin G instead of the rela- 
tively ineffective penicillin K. On the other hand, the constant promise that penicillin 
would be produced synthetically and make fermentation obsolete, was a real deterrent 
for some companies to invest in fermentation facilities. If it had not been for the sense 
of urgency created by the war, and thus the push to produce all the penicillin possible 
by the best existing techniques, the rapid advances in fermentation technology might 
have been significantly delayed in anticipation of a breakthrough in penicillin synthesis. 



E-9 
REFERENCES 

Abraham, E. P., Chain, E. , Fletcher, C. M. , Florey, H. W. , Gardner, A. D, , 
Heatley, N. G. , and Jennings, M. A., Lancet, 2, 177(1941). 

Backus, M. P., and Stauffer, J. F. , Mycologia, XLVII, 429 (1955). 

Chain, E. , Florey, H. W. , Gardner, A. D. , Heatley, N. G. , Jennings, M. A., Orr- 
Ewing, J., and Sanders, A. G. , Lancet, 2_, 226 (1940). 

Clark, H. T. ( ed. ), The Chemistry of Penicillin, Princeton University Press, New 
Jersey (1949). 

Clutterbuck, P. W. , Lovell, R. , and Raistrick, H. , Biochem. J., 26, 1907(1932). 

Coghill, R. D. , Chem. Eng. News, 22, 588 (1944). 

Dubos, R. J., Proc. Soc. Exptl. Biol. Med., 40, 3 1 1 (1939a). 

Dubos, R. J., J. Exper. Med., 70, 1 (1939b). 

Dubos, R, J., J. Exper. Med., 7^, 11 (1939c). 

Elder, A. E. , The History of Penicillin Production, AIChE Chem. Eng. Prog. Symp. 
Ser. No. 100 (1970). 

Emmerich, R. , and Low, O., Ztschr. f. Hyg. u. Immunitat. , 3 1, 1 (1899). 

Fleming, A., Proc. Roy. Soc, B, XClll , 306 (1922). 

Fleming, A., Brit. J. Exp. Path., H), 226(1929). 

Fleming, A., J. Path. & Bact. , 35, 83 1 (1932). 

Florey, H. W. , and Jennings, M. A., Brit. J. Exp. Path., 23_, 120(1942). 

Florey, M. E. , and Florey, H. W. , Lancet, J_, 387(1943). 

Foster, J. W. , and Woodruff, H. B. , J. Bact., 46, 187(1943). 

Goris, A., and Liot, A., C. R. Acad. Sci. , 172 , 1622(1921). 

Gratia, A., and Dath, S. , Compt. rend. Soc. de Biol., 92, 461 (1925). 

Hare, R., The Birth of Penicillin, and the Disarming of Microbes , George Allen and 
Unwin Ltd., London (1970). 

Hobby, G. L. , Meyer, K. , and Chaffee, E. , Proc. Soc. Exp. Biol. & Med., 5_0, 277 
(1942a). 

Hobby, G. L. , Meyer, K. , and Chaffee, E. , Proc. Soc. Exp. Biol. & Med., 50, 285 
(1942b). 

Joslyn, D. A., Science, 99, 21 (1944). 



E-10 

Keefer, C. S. , Blake, F. G. , Marshall, E. K. , Jr., Lockwood, J. S. , and Wood, 
W. B. , Jr., JAMA, 122, 1217(1943). 

Lee, S. W. , Foley, E. J., Epstein, J. A., and Wallace, J. H. , Jr., J. Biol. Chem. , 
152 , 485 (1944). 

Lyons, C, JAMA, 123 , 1007(1943). 

Mahoney, J. F. , Arnold, R. D. , and Harris, A. D, , Am. J. Pub. Health, 33_, 1387 
(1943). 

McAdam, I. W.J., Duguid, J. P., and Challinor, S. W. , Lancet, 2, 336(1944). 

McMahan, J. R. , J. Biol. Chem., 152_, 249 (1944). 

Moyer, A. J,, and Coghill, R. D. , J. Bact. , 5_1_, 57 (1946a). 

Moyer, A. J., and Coghill, R. D. , J. Bact., 5j_, 79 (1946b). 

Moyer, A. J., and Coghill, R. D. , J. Bact., 53^, 329 (1947). 

Pasteur, L. , and Joubert, C. R. , C. R. Acad. Sci. Paris, 85_, 101(1877). 

Pratt, R. , and Hok, K, A., Am. J. Bot. , 33_, 149 (1946). 

Rammelkamp, C. H. , and Keefer, C. S. , J. Clin. Invest., 22, 425 (1943), 

Raper, K, B. , U. S. Dept. Agr. Yearbook, 699 (1943). 

Raper, K. B. , personal communication ( 1975). 

Raper, K. B. , and Alexander, D. F. , Elisha Mitchell Sci. Soc. J., 6_]_, 74(1945). 

Raper, K. B. , Alexander, D. F. , and Coghill, R. D. , J. Bact., 48, 639(1944). 

Reid, R. D. , J. Bact., Z5_, 31 (1933). 

Reid, R. D. , J. Bact., 29^, 215(1935). 

Richards, A. N. , Nature, 201 , 441 (1964). 

Robinson, H. J., J. Pharmacol. & Exp. Therap. , 77, 70(1943). 

Schatz, A. , Bugie, E. , and Waksman, S. A. , Proc. Soc. Exp. Biol. N. Y. , 55, 66 
(>944). 

Schmidt, L. H. , and Moyer, A. J., J. Bact., 47, 199(1944). 

Waksman, S. A., and Woodruff, H. B. , Proc. Soc. Exp. Biol, k Med., 45, 609 (1940). 

Weindling, R. , and Emerson, O. H. , Phytopath. , 26, 1068(1936). 

Wells, P. A., Lockwood, L. B. , Stubbs, J. J., Roe, E. T. , Porges, N. , and 
Gastrock, E. A.. Ind. & Eng. Chem., 34, 1518(1939). 



r 



ANTIBACTERIAL MATERIALS 
FROM IVIICROORGANISMS 



. 1877 Pasleur Eltc 



Qllect on oihGi bacle 
NB/S/W/ / 



r 



1925 Catiit Moldi • 



DISCOVERY AND 
CHARACTERISTICS 



s Diicovers baciericidsl Bgeni 
(tv'olh'icln) produced by so 



NR/S/U/G/GL 



1929 Fleming Ditc 



1932 Fleming RepofH 



— 1932 Clunetbuck Pani< 



uflectivc 
cKdurc lor penicillin deicriticd 
NR/S/W/ /M 

-1933 Clulieibuck Df. Thorn ol USDA cti'iBCll^ 
Idoniilics FJcming'i mold at 
Penieillium nolaium Westling 

NR/D/U/G/GL 

- 1933 Reid Dcmonsiralct Iho uniqueneii ol the 
Pcnicillla moldi in produ'ting iho 
inhibitory subilano) 

NR/S/U/G/M 



CODES 

Nonmission-Oriented Research 

Mission-Orienied Research 

Deuelopmenl 

Sigriificani Event 

Decisive Eveni 

U.S. Event 

Foreign Event 

Government Funding 

Private Funding 

Medical School - Hospital — 

University 

Research Institution 

Government Labotatory 

Company 



- 1940 Chain Chemotheisi 
sniitisll 



'LY COME TO UNITED STATES 



Mfl/S/W/G/M 

1942 Hobby QheinuiheiopQutic sctiviiv o 
MR/S/U/P/M 



1913 Robinson (Donfli™ low loxleiiy ot 



- FERMENTATION TECHNOLOGY 



!i Produclion capabilil 



ate developed 

DV/S/U/G/GL 



— HUMAN CLINICAL TRIALS 



1941 Abraham IXic 



1946 Woyci Achievei 100-fold incri 
(1942- yield of penldllln by it 

194S] of oisjnijm, uw of cor 



irie of the feimcntBli 
DV/D/U/G/GL 



- 1946 Mov*r Peni. 
M942- irarr 

19451 



DV/D/U/G/GL 



1942 MERCK AND CO., INC.. E. R. SQUI8B 
AND SONS, AND CHAS. PFIZER AND 
CO. FORMALLY AGFieE TO COL- 
LABORATE ON PENICILLIN 
PROGRAM 



MR/D/W/G,P/M 



B chamolhcrapGu 



MR/S/U/G/GL 



ASSAY METHODS 

Foiicr Modification ol cup aiiay 

DV/S/U/P/C 

Schmidt Hollnoment ol the cylindei 
plalo ntolJiod for oonkillin 



1 Oevelopi impiovcd lurbid 
a>iDy procedure toi panic 
DV/S/U/P/C 



1 procedure lor penicilli 
dovBtopcd 

DV/S/U/P/C 



J 



HISTORIOGRAPH - PENICILLIN 



1955 


eeckui Hi 


hly productive -train 


11945 




w 


. QI76 developed 
DV/S/U/P/M 


1946 


Pratt 


Ropo 










balonec at inoiganic 






















yicUh 


of penicillin 
DV/S/U/P/C 



1943 Lyons Reportj an 209 caiM ol pt 
DV/D/U/G/M 



.1944 McAdam Uic o 



L 



MR/S/W/P/M 
MARCH. 1945 COMMERCIAL DISTRIBUTION BEGINS 



F-1 



APPENDIX F. L-DOPA 



INTRODUCTION AND SUMMARY 



U. S. Food and Drug Administration (FDA) approval of L-dopa in 1970 signaled a 
new era in the treatment of neurological disease. General acceptance of this drug as the 
most effective agent in the treatment of parkinsonism is highly significant in itself. But 
the precedent set by the scientific basis for L-dopa therapy may have more universal 
pharmacological impact. 

Prior to the initial clinical trials with L-dopa in Parkinson's disease, neurological 
disorders of this type were treated surgically or empirically with drugs. Scientific 
advances made in the 1950's resulted in (1) the general appreciation of the role of the 
catecholamine as neurotransmitters and (2) the elucidation of the catecholamine biosyn- 
thetic pathway. In I960, the crucial observation was made that parkinsonism was 
characterized by abnormal catecholamine (especially dopamine) metabolism. Clinical 
trials in parkinsonian patients using dopa, the in vivo precursor of dopamine, quickly 
suggested the benefits of this type of "replacement therapy" for neurological disorders. 

Subsequent clinical trials, however, indicated that the dopa therapy used in early 
clinical trials was ineffective, and by 1966, the use of this drug ■was still generally 
unproven. 

The dramatic discovery that led to final FDA release of L-dopa was the introduction 
of a radical method of drug administration to give long-lasting alleviation of parkinsonian 
symptoms in most patients. In 1967, the necessity for gradual development of tolerance 
to large doses of the drug was finally accepted. 

The final impetus for rapid FDA approval of L-dopa was provided by competition 
between two drug companies. Large, multicenter clinical studies were initiated in 1969 
by these companies and the efficacious usage of L-dopa was generally substantiated early 
in 1970. 

Since L-dopa was released for clinical use, investigations have continued into the 
role of this drug in catecholamiine neurophysiology and into optimizing the therapeutic 
regimen both in parkinsonism and related diseases. There is little doubt, however, con- 
cerning the significant effect of the introduction of L-dopa in the treatment of parkinson- 
ism and in fostering the scientific approach to the treatment of other neurological 
disorders. 



HISTORICAL ACCOUNT 



The neurological disease bearing his name was first described by James Parkinson 
in 1817 and is characterized symptomatically by akinesia, rigidity, tremor, and dis- 
equilibrium. In the years preceding the acceptance of L-dopa therapy, parkinsonism was 
treated surgically or with anticholinergic or antihistaminic drugs. It is important in the 
present context to recognize that these approaches represented an empirical treatment of 
the symptoms of parkinsonism without a rational basis in the neurological disorders 
underlying the disease. 



F-2 

The insights which precipitated the clinical administration of L-dopa in parkin- 
sonism, however, led logically to this therapy based on knowledge of the associated 
neurophysiology and catechol metabolism, as shown on the historiograph. 



Catecholamine Neurophysiology 



The current understanding of neurotransmission originated with the experiments of 
Elliott in England in 1905. He observed that injection of epinephrine in aniraals resulted 
in a response similar to that produced by electrical stimulation of sympathetic nerves. 
This finding led to the postulation that an epinephrine -1 ike substance might be released 
by the sympathetic nerve and stimulate the adjoining muscle. The first direct experi- 
mental evidence for chemical transmission was given by Loewi in Austria in 1921 in the 
parasympathetic system. 

It was not until 1946 that von Euler in Sweden positively identified norepinephrine as 
a neurotransmitter of the sympathetic system. This slow development of understanding 
of the physiology of the sympathetic system was due, at least in part, to the difficulties 
involved in detecting, isolating, and quantifying the catecholamines involved. These 
difficulties were responsible for the fact that dopamine detection was not reliably per- 
formed in the brain until 1957 and is not a simple matter even today. Advances in 
chromatographic separations and spectrofluorimetry, to a large part due to the work of 
Axelrod, Udenfriend, Brodie, and others at the National Institutes of Health, eventually 
led to sensitive methods for catecholamine analysis. 

Thus, in 1951 when Raab and Gigee found a catecholamine with epinephrine-like 
activity concentrated in the mammalian caudate nucleus, which could be increased in 
concentration by I. P. injection of L-dopa, they -were unable to identify positively the 
compound as dopamine. (They called the compound "encephalin". ) Not until 1957 was 
dopamine tentatively identified in brain homogenates by Montagu and then positively 
identified by Carlsson and his associates. 

In the meantime, Vogt (1954) had determined the distribution of norepinephrine in 
the brain which indicated the differences in function of the brain regions. 

The period from 1957 to 1959 is marked by the contributions of Carlsson and his 
associates in Sweden. On the basis of the principles of spectrofluorimetry learned in the 
United States, these researchers developed sensitive methods for detecting dopamine. 
They then found that reserpine, an antihypertensive agent capable of inducing parkinsonian 
symptoms, depleted the brain of dopamine (Carlsson, Bertler, et al. , 1957). In a series 
of crucial experiments, they then found that dopa could counteract the extrapyramidal 
symptoms associated with reserpine injection, while the serotonin precursor, 
5-hydroxytryptophan, did not (Carlsson, Lindqvist, and Magnusson, 1957). They soon 
learned that L-dopa also increased brain dopamine levels in reserpinized animals 
(Carlsson, et al. , 1958). These experiments thus set the precedent for L-dopa therapy 
in pharmacological parkinsonism. 

Carlsson and his associates subsequently determined the regional distribution of 
dopamine in the brain, finding high concentrations in the caudate nucleus and striatum. 
They proposed that dopamine may be involved in the extrapyramidal control of motor 
functions (Carlsson, 1959; Bertler and Rosengren, 1959). 



F-3 

At nearly the same time, researchers in Japan had studied the regional distribu- 
tion of dopamine, norepinephrine, and dopa in human brains obtained 10 to 20 hours 
post mortem (Sano, et al. , 1959). They found essentially the same regional distribu- 
tions as the workers in Sweden and also noted that since the dopamine and norepinephrine 
distributions differed, then dopamine may have a function other than simply as the 
norephinephrine precursor. Perhaps more importantly, these experiments showed that 
the brain catecholamine distributions were not rapidly altered post mortem. This 
finding permitted analysis of post mortem brain material to determine disease etiology 
at the neurotransmittor level. 

By I960, therefore, the elucidation of catecholamine neurophysiology had progressed 
to the point that the search for abnormal catecholamine distributions in neurological 
disease was a logical approach. The crucial experiments made in Parkinson's disease 
by Ehringer and Hornykiewicz in Vienna in I960 and by other workers is described below. 

Since I960, work in the area of neurotransmission has accelerated rapidly. At the 
present time, it seems reasonable to say that the mechanisms of sympathetic neuro- 
communication are basically understood (Axelrod, 1974). On the historiograph, there 
has been no attempt to indicate all of the significant events which occurred after 1960 
since they were numerous and did not contribute directly to the introduction of L-dopa as 
a drug. Rather, the events shown are n:ieant to serve as markers of this progress. 

The development in the early 196 0's of fluorescent-histochemical methods for the 
detection of biogenic amines permitted sensitive determination of catecholamine dis- 
tributions at the cellular level (Falck, et al. , 1962). These methods, also made by 
associates of Carlsson in Sweden, eventually led to the present understanding of the 
structure and function of the sympathetic nerve cells (Axelrod, 1974). 

Advances in analytical methodology also resulted in the mapping of catecholamine 
metabolism in the brain. Thus, the O-methylated product resulting from dopamine 
metabolism, homovanillic acid, was soon found to parallel dopamine in its distribution 
(Sharman, 1963; Bernheimer, 1.964). 

By 1970, the mapping of brain catecholamine metabolism had advanced to the 
enzyme level with the determination of the regional distribution of the dopamine synthe- 
sizing enzymes (Cote and Fahn, 1969; Lloyd and Hornykiewicz, 1970; Vogel, et al. , 1969). 

Another indication of the impressive developnnents in the understanding of neuro- 
transmission was the awarding of the 1970 Nobel prize for medicine or physiology which 
was shared by von Euler, Axelrod, and Katz (Udenfriend and Martin, 1970). 



Catecholamine Metabolism 

Although the elucidation of the catecholamine biosynthetic pathway is generally not 
emphasized in the physiological or clinical literature, it seems important in the present 
context. 

The first postulation of the correct catecholamine biosynthetic pathway, starting 
with tyrosine, was made by Blaschko in 1939 to be 

tyrosine *- dopa >■ dopamine >- norepinephrine >- epinephrine. 



F-4 

Although the biosynthetic pathway has been found to be more complex than 
described here (Molinoff and Axelrod, 1971; Cotzias, et al. , 1971), this was the accepted 
pathway around I96 0. 

Holtz was the first to give in vivo evidence for transformation of dopa to dopamine 
in 194Z. Subsequent elucidation of the enzymes involved took place from the early 1950's, 
largely in the laboratories of the National Institutes of Health under the direction of 
Udenfriend, Brodie, and Axelrod. These experiments culminated in the isolation of the 
enzymes of the peripheral sympathetic nervous system in the early 1960's. 

Even without detailed understanding of the enzymes involved, it was probably the 
general realization that dopa is involved in catecholamine biosynthesis which prompted 
Raab and Gigee (1951) to inject L-dopa in order to note the effect on "encephaline" 
concentration. In the same way, when Carlsson used dopa to antagonize the reserpine- 
induced parkinsonian syndrome, it was with the appreciation that dopa was the dopamine 
precursor. 



L-Dopa Therapy for Parkinsonism 

The line of research leading to effective treatment of Parkinson's disease with 
L-dopa emanated from the now-classic studies of Ehringer and Hornykiewicz in Vienna 
in I960. By comparison of the distribution of norepinephrine and dopamine in normal 
and parkinsonian human brains post mortem, they concluded that parkinsonism is 
characterized by significant depletion of the dopamine in the neostriatum. Thus, the 
connection between symptomatic neurological disease and chemical neurotransmittors 
was established. 

A factor which probably accelerated this discovery was the supply of post-mortem 
brain material from parkinsonian patients available to these researchers. At the time, 
autopsies were performed on nearly all patients who died in Austrian hospitals, as 
required by law. Thus, Ehringer and Hornykiewicz were provided with a steady supply 
of diseased brains, and the necessity for coordinated procedures for collecting specimens 
vi'as eliminated. 

Following the experiments by Carlsson and his associates in Sweden in 1957 in 
reserpinized mice, clinical trials of L-dopa advanced rapidly to humans. In I960, 
Degkwitz and co-workers in Germany first administered L-dopa to humans alone and 
after injection of several drugs including reserpine. In 1961, McGeer and associates in 
Canada treated patients with pharmacological parkinsonism induced by reserpine or 
phenothiazines. Improvement in extrapyramidal symptoms was noted in two of four 
reserpinized patients given D, L-dopa, but in general, dopa therapy was not recommended 
for treatment of drug-induced parkinsonism. 

The realization that Parkinson's disease v^as characterized by decreased dopamine 
levels quickly led to clinical attempts to alleviate this symptom through "replacement 
therapy". It was generally felt at the time that dopamine could not readily penetrate the 
blood-brain barrier. Thus, L-dopa, the dopamine precursor which antagonized reser- 
pine, was utilized in the first clinical trials in parkinsonism. 

Although the exact sequence of events is difficult to reconstruct, it appears that 
tv70 groups independently tried L-dopa therapy nearly simultaneously in 1961. In Vienna, 
Birkmayer and Hornykiewicz injected L-dopa intravenously and reported dramatic but 



F-5 

short-lived alleviation of parkinsonian akinesia. Barbeau in Montreal administered 
L-dopa orally and described equally impressive effects on symptomatic rigidity and 
tremor. The step between (1) recognition of the association of depleted dopamine levels 
and Parkinson's disease and (2) clinical trial of a rational replacement therapy was 
therefore achieved in less than a year. 

Following the initial successes reported with L-dopa therapy, there occurred a 
4- or 5-year period of clinical trials with the drug. Many dosages and combinations of 
drugs were administered both orally and intravenously. Generally speaking, the early 
trials were considered "favorable" in terms of alleviation of parkinsonian symptoms. 
Evidence soon began to accumulate, however, which questioned the validity of the 
previously reported successes (e.g., McGeer and Zeldowicz, 1964; Duvoisin, 1965). 
When two double-blind studies indicated negative results with L-dopa (Fehling, 1966; 
Rinne and Sonninen, 1968), the initial enthusiasm for L-dopa therapy had nearly 
disappeared. 

During the period from 1961-1966, many factors probably contributed to retard 
and eventually discredit the use of L-dopa. For example, no companies were producing 
L-dopa in quantity. Consequently, the drug v^as sufficiently scarce and expensive to 
discourage prolonged administration of the "massive" quantities (based on pharmacologi- 
cal practice at the time) which were eventually shown to be required. The need for 
frequent doses on a long-term basis also argued against the intravenous delivery route. 

None of the pharmaceutical companies or governments active in this field were 
moving aggressively to test the efficacy of the new therapy. Although Hoffmann- LaRoche 
(especially in Europe) was instrumental in producing and distributing small quantities of 
L-dopa in the early 1960's, the company was not making a concerted effort to gain the 
drug's approval. Since the drug was considered to be unpatentable and was directed 
towards a relatively small market, the lack of commercial interest is understandable. 

It is more difficult to determine the factors which contributed to attitudes within 
the group of clinical investigators actually using the drug. In retrospect, there seems 
to be substantial doubt that the early clinical trials of L-dopa had shown any real 
improvements in the alleviation of parkinsonism. This is supported by many subsequent 
observations that the appearance of abnormal involuntary movements which were 
eventually associated with successful L-dopa therapy were never reported in the early 
studies. The lack of controlled clinical evaluations surely slowed the search for the 
optimum delivery method for the drug. For whatever reasons, however, it seems clear 
that the methods used clinically until 1966 were ineffective in producing the optimum 
regimen for L-dopa therapy. 

In the middle 1960's, Ajinomoto Company in Japan began to produce dopa by fer- 
mentation and to distribute the material in the United States through the Nutritional 
Biochemical Company. With the availability of large quantities of the drug, Cotzias and 
his associates at the Brookhaven National Laboratories, with the support of the Atomic 
Energy Commission and the National Institutes of Health, began clinical trials in 1966 
which eventually led to the approval of L-dopa. 

Cotzias and his associates had previously been interested in the interconnection of 
biogenic amines and manganese in neurological diseases (Cotzias, et al. , 1964). For 
many years these ■workers had been active in determining the physiological role of trace 
metals. They had developed and employed sensitive analytical methods for manganese 
determination based on neutron activation analyses and radioisotopic techniques. Cotzias 
and his co-workers recognized that some extrapyramidal symptoms displayed in 



F-6 

chronic manganese poisoning were also associated with parkinsonism. Furthermore, 
they realized that observations of depigmentation of certain brain areas in parkinsonism 
were probably related to low numbers of melanin granules, which were known to contain 
many trace elements, including manganese. Melanin was believed to be a dark pigment 
formed by polymerization of quinones or semiquinones following oxidation of dopa 
(Cotzias, et al. , 1964). Thus, Cotzias and his co-workers entered the field of Parkin- 
son's disease with somewhat different interests than most other active groups, and with 
perhaps a greater appreciation of the complexities of L-dopa metabolism. 

Initial attempts to increase melanin content in parkinsonian patients with the 
melanophore -stimulating hormone /3-MSH increased the associated tremor. Melatonin, 
which antagonizes certain actions of /3-MSH, controlled the tremor but caused sedation. 
Experiments were then switched to melanin precursors. Phenylalanine was either 
ineffective or also aggravated the symptoms, so Cotzias and his associates began 
administering dopa. They decided to try to saturate the enzymes acting on dopa by 
slowly increasing daily doses of the drug. This finally resulted in the key observations 
leading to effective L-dopa therapy in parkinsonism (Cotzias, et al. , 1967). In a 
collaborative effort between Cotzias, Mena, and their associates, this therapy was also 
shown to be effective in the treatment of Chilean patients with chronic manganesism 
(Mena, et al. , 1970). 

By 1967, Cotzias and his co-workers had demonstrated successful long-term 
treatment of parkinsonian patients with D, L-dopa using a novel regimen which included 
the following important features: 

(1) The necessity for slovirly developing patient tolerance for the drug by 
increasing the oral dosage every 2 or 3 days in 0. 5-gram increments 
or at a rate which did not provoke significant side effects 

(2) The final attainment of very high daily dosages (up to 16 grams per 
day), the final level being adjusted on an individual-patient basis to 
give the maximum symptomatic relief while minimizing untoward 
side effects 

(3) The need to maintain this therapy for prolonged periods ( 1 to 2 years) 
in order to achieve greater amelioration of symptoms. 

The dramatic successes of Cotzias' trials were illustrated at several international 
meetings, such as the Third Parkinsonism Symposium in Edinburg in May, 1968, and 
these quickly led to a resurgence of interest in the drug. Several well-controlled trials 
(notably that of Yahr, et al. , 1969) soon verified the essential aspects of Cotzias' s 
findings. In later trials with L-dopa, Cotzias and his associates (1969) showed that the 
total dosage of dopa could essentially be halved to a maximum of about 8 grams per day 
in extreme cases with reduction of many of the side effects of D, L-dopa therapy. 

Interest in the new treatment grew at Eaton Laboratories and at Hoffmann- LaRoche, 
which soon led to large, multicentered clinical trials with L-dopa (Keenan, 1970; 
Langrall and Joseph, 1971) with essentially the same findings as the earlier studies. 

In June, 1970, under growing pressure from the pharmaceutical industry, clinicians, 
and the general public, the FDA released the drug on a limited basis to neurological 
clinics and hospitals affiliated with medical schools and to clinical investigators. 

For his contributions to medical therapy in neurological disease, Dr. Cotzias 
received the Albert Lasker Clinical Medical Research Award in 1969. 



F-7 

L-Dopa Potentiators 

In the early 1960's, several hydrazine inhibitors of aromatic amino acid decarboxy- 
lase were developed for use in arterial hypertension (Pletscher, et al. , 1970). Although 
these derivatives were found ineffective in this application, they have been successfully 
incorporated into L-dopa therapy. 

In 1966, Udenfriend and associates at the National Heart Institute observed that 
a-methyldopa hydrazine (Merck, Sharp, & Dohme # MK-485) enhanced the conversion of 
dopa to catecholamines in the brain and inhibited the extracerebral decarboxylases. 

Birkmayer (1967) and Birkmayer and Mentasti (1967) soon reported experiments 
which indicated enhancement of the effect of L-dopa in parkinsonism when another 
decarboxylase inhibitor (Hoffmann-LaRoche, Ro4-4602) was administered along with 
L-dopa. Soon thereafter, Bartholini and co-workers (1967) at Hoffmann-LaRoche 
observed that Ro4-4602 in low doses selectively inhibits extracerebral decarboxylase 
activity. Thus, the concentration of I. P. -injected L-dopa is increased in the blood and 
finds its way to the brain in greater quantities. They also suggested treatment of par- 
kinsonism with combinations of L-dopa and decarboxylase inhibitors. 

In 1969, Cotzias and co-workers reported that coadministration of L-dopa and 
L-a-methyldopahydrazine reduced the total dosage required and eliminated some of the 
side effects of L-dopa therapy. 

At about the same time, Duvoisin and his associates observed that pyridoxine 
(Vitanain Bz), taken along with L-dopa, can decrease the effectiveness of the drug. It is 
possible that pyridoxine, administered along with L-dopa in the early clinical trials of 
the drug, may have contributed to the uncertainty surrounding the effects of L-dopa in 
parkinsonism. This inhibition by pyridoxine also led to marketing of a pyridoxine-free 
vitamin preparation (Cotzias, 1972). By 1972, Cotzias' s group had shown that L-a- 
methyldopahydrazine not only decreased the dosage of L-dopa required and permitted 
more rapid induction of therapy, but also allowed coadministration of pyridoxine 
(Papavasiliou, 1972). 

Because of these findings and owing to this potentiator' s low toxicity, L-a- 
methyldopahydrazine was approved by the FDA in 1975 and is now marketed in combina- 
tion with L-dopa by Merck, Sharp, &: Dohme. 



Related Developments 

The brief discussion above was intended to describe the factors and events which 
contributed directly to the discovery and general availability of L-dopa. Unfortunately, 
a surmmary of this type fails to completely describe the far-reaching pharmacological 
impact of this therapy. 

For example, clinical trials of L-dopa in parkinsonism, based on the dopamine - 
replacement concept, stimulated trials of the drug in many other neurological diseases. 
Therapy using L-dopa has also resulted in improved detection of various forms of par- 
kinsonism. Besides parkinsonism and chronic manganese poisoning, for example, 
L-dopa has also been tried with varying success in torsion dystonia, progressive supra- 
nuclear palsy, hepatic coma, depression, and many other disorders, and as a provoca- 
tive agent for Huntington's chorea (see Pelton and Chase, 1975, for a recent review). 



F-8 

The success with L-dopa also added impetus to the tendency toward scientific input 
into neuropharmacology. Thus, for example, parkinsonism research is continuing in the 
development of dopamine receptor agonists (activators) as opposed to dopamiine pre- 
cursors such as L-dopa (Cotzias, 1975). There is also an increasing recognition that 
neurological diseases are often characterized by problems in both adrenergic and 
cholinergic systems, leading to a revival of anticholinergic drugs in combination with 
L-dopa. 

Finally, there is a growing awareness that the treatment of parkinsonism by 
replacing deficient dopamine pools still represents only syinptomatic treatment of the 
disease, albeit at the molecular level. Thus, the stage is set for many exciting dis- 
coveries in the etiology of neurological disease. 



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Bernheimer, H. , Nature, 204, 587(1964). 

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Birkmayer, W. , and Mentasti, M. , Arch. Psychiat. Nervenkr. , 210 , 29 (1967). 

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Carlsson, A., Pharmacol. Rev., n_, 490(1959). 

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(1958). 

Carlsson, A,, Rosengren, E. , Bertler, A., andNilsson, J., in Psychotropic Drugs, 
S. Garattini and V. Ghetti (Eds.), Elsevier Publishing Co., Amsterdam, 363 (1957). 

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Brunette (Eds.), Exerpta Medica Foundation, Amsterdam, 311 (1969). 

Cotzias, G. L. , New Eng. J. Med., 278 , 630(1968). 

Cotzias, G. C. , Harvey Lectures, 115 (1972-73). 



F-9 

Cotzias, G. C. , in Proceedings of the 1975 Annual Meeting, Association for Research 
in Nervous and Mental Disease, New York, December 5-6, 1975 (in press). 

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(1969). 

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Med. , 22, 305 (1971). 

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Proc. , 23, Parti, 713(1964). 

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374 (1967). 

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38(3), 120 (I960). 

Duvoisin, R. C. , Bull. N. Y. Acad. Med., 41_, 898(1965). 

Duvoisin, R. C. , Yahr, M. D. , and Cote, L. D. , Trans. Amer. Neurol. Assn. , 94, 
81 (1969). ~ 

Ehringer, H. , and Hornykiewicz, O. , Klin. Wochschr. , 38, 1236 (I960). 

Elliott, T. R. , J. Physiol. London, 32, 401 (1905). 

Falck, B. , Hillarp, N. A., Thieme, G. , and Torp, A., J. Histochem. Cytochem. , 10, 
348 (1962). 

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(1942). 

Keenan, R. E., Neurology, 20(12), Part 2, 46 (1970). 

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Leeper, L. C. , and Udenfriend, S, , Fed. Proc, j_5, 298(1956). 

Lloyd, K. , and Hornykiewicz, O. , Science, 170 , 1212 (1970). 

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Lovenberg, W. , Weissbach, H. , and Udenfriend, S. , J. Biol. Chem. , 237(1), 89(1962). 

McGeer, P. L. , Boulding, J. E. , Gibson, W. C. , andFoulkes, R. G. , JAMA, 177(10), 
665 (1961). 

McGeer, P. L. , and Zeldowicz, L. R. , Canad. Med. Assoc. J,, 90, 463 (1964). 

Mena, I. , Court, J. , Fuenzalida, S. , Papavasiliou, P. S. , and Cotzias, G. C. , New 
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F-10 

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Papavasiliou, P. S. , Cotzias, G. C. , Diiby, S. E. , Stack, A. J., Fehling, C. , and 
Bell, M. A., New Eng. J. Med., 285 , 8 (1972). 

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S. Garattini, A. Goldin, F. Hawking, and I. J. Kopin (Eds.), Academic Press, New 
York, 253 (1975). 

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Biochem. Biophys. Acta, 32, 586 (1959). 

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Udenfriend, S. , Cooper, J. R. , Clark, C. T. , and Baer, J. E. , Science, 117, 663 
(1953). 

Udenfriend, S. , and Martin, A. R. , Science, 170 , 422 (1970). 

Udenfriend, S. , and Wyngaarden, J. B. , Biochim. Biophys. Acta, 20, 48 (1956). 

Udenfriend, S. , Zaltzman-Nirenberg, P. , Gordon, R. , and Spector, S. , Mol. 
Pharmacol., 2, 95 (1966). 

Vogel, W. H. , Orfei, V., and Century, B. , J. Pharmacol. Exp. Therap., 165 (2), 196 
(1969). 

Vogt, M. , J. Physiol., 123 , 451 (1954). 

von Euler, U. S. , Acta Physiol. Scand. , J_2, 73 (1946). 

Yahr, M. D. , Duvoisin, R. C. , Schear, M. J. , Barrett, R. E. , and Hoehn, M. M. , 
Arch. Neurol., 21, 343 (1969). 



CATECHOLAMINE NEUROPHYSIOLOGY 



1905 Elliott First poslulation of chemical 
transmission at synapses by 
epinephrine-like substance 



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CATECHOLAMINE NEUROPHYSIOLOGY 




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HISTORIOGRAPH - L-DOPA THERAPY For Parkinson's Dist 



G-1 
APPENDIX G. PHENOTHIAZINES 

INTRODUCTION AND SUMMARY 



The introduction of phenothiazine derivatives into clinical medicine for the treat- 
ment of (1) psychiatric patients, (2) nausea and vomiting, (3) allergies, (4) parkinson- 
ism, and (5) pruritus represents one of the great technological interdisciplinary 
triumphs of the chemical, biological, medical, and societal sciences. Of the pheno- 
thiazine derivatives, the impact of chlorpromazine (CPZ) in psychiatry greatly 
exceeded the imagination of those who initially created and evaluated it. More than 
100, 000, 000 people have been treated with CPZ and the number of publications con- 
cerning it approximates 15, 000. 

This case study delineates the discovery, evaluation, and therapeutic significance 
of the phenothiazine s with emphasis on CPZ's effects against psychiatric diseases, 
particularly schizophrenia. Described also is CPZ's role in changing the public mental 
hospital from a custodial institution to a therapeutic one and CPZ's influence in creating 
a favorable environment for outpatient care. Consideration is also given to CPZ's role 
in changing the ideologies of mental-health personnel concerning drug therapy and in 
altering lay attitudes toward mental disorders and the mentally ill. CPZ's economic 
importance for the pharmaceutical industry, for mental patients, and for patients able 
to regain normalcy in society because of maintenance drug therapy is discussed. 
Examined also are the feedback effects of chlorpromazine on basic and clinical research 
and the stimulus they provided for (1) the development of new psychotropic drugs, 
(2) new methodology for their evaluation, and (3) the emergence of psychopharmacology 
as a discipline in its own right. 

The first clinical tests on CPZ in the United States were initiated in 1952. The 
original synthesis of CPZ by Paul Charpentier and the pharmacological assays by 
Madame S. Courvoisier occurred at the Rhone-Poulenc Laboratories in France in 1950. 
CPZ's building block, phenothiazine, had been synthesized by Bernsthen in Germany in 
1883. Prefacing the synthesis of phenothiazine was a series of discoveries and devel- 
opments in structural, organic, and physical chemistry, beginning with Faraday's iso- 
lation of benzene in 1825. Laborit's work in 1949 on the use of synthetic antihistamines, 
especially promethazine, in surgery to combat shock, and earlier observations by 
Sigwald, et al. , in 1956 on the antiparkinsonian effects of fenethazine, promethazine, 
and diethazine, not only confirmed the value of these materials as antihistaminics and 
antiparkinsonian drugs, but also revealed their clinical sedative character, a property 
that strongly influenced Rhone -Poulenc 's decision in 1950 to begin the research that 
produced chlorpromazine. By 1970, pharmaceutical manufacturers in the United States 
had shipped over $100 million worth of CPZ and other phenothiazine derivatives to be 
used by about 35 percent of adult Americans in combating mental illness, a disease 
whose tangible and intangible costs approximate $20 billion per year. 



HISTORICAL ACCOUNT 

The historical account is divided into sections on (1) synthesis of phenothiazine, 
(2) search for synthetic antihistaminic drugs, (3) synthesis and evaluation of chlor- 
promazine, and (4) chlorpromazine in the United States. For each section, key 
chronological events will be discussed and the historiograph will illustrate events 
chronologically. 



G-2 

Synthesis of Phenothiazine 

The synthesis of phenothiazine by Bernsthen in 1883 and the synthesis of CPZ by 
Charpentier, et al. , in 1950 were prefaced by two greatly different periods of develop- 
ment in the chemical and biological fields. Benzene was discovered and the charac- 
terization of benzene and its homologues, which ushered in the dye industry during the 
last half of the 19th century, was initiated during the first half of the 19th century. 
Faraday discovered benzene in compressed gas in 1825. Its commercial availability 
was assured when Mansfield in 1848 isolated substantially pure benzene, toluene, and 
pseudocumene from coal tar by fractional distillation. In 1934, Mitscherlich converted 
benzoic acid into benzene by dry distillation with lime. The benzene was converted to 
nitrobenzene using a mixture of sodium nitrate and sulfuric acid. A large-scale plant 
for the preparation of nitrobenzene using sodium nitrate and sulfuric acid was con- 
structed by Perkins in England in 1857. Cannizzaro's work, published in 1858, helped 
to clarify anomalies between atomic and molecular weights of organic compounds. 
Kekule's resonance theory for benzene, which he announced in 1865, found general 
application to many organic chemicals found in coal tar. Beginning with Unverdorben 
in 1826, a number of investigators prepared "anilines" from coal-tar sources, by the 
dry distillation of indigo, or by the reduction of nitrobenzene. It was Hoffmann who, 
in 1843, proved that all the "anilines" were the same. Bechamp's preparation of 
aniline in 1853 by the reduction of nitrobenzene with iron and acetic acid paved the way 
for the production of aromatic amines, key materials in the dye industry. In 1856, 
Perkin oxidized a dilute solution of crude aniline sulfate with potassium dichromate to 
obtain about 5 percent of a violet dye which was manufactured as mauve. Perkin 's dis- 
covery gave great impetus to the research on aniline as a basis for synthetic dyes. 
Ironically, research on mauve and other dyes produced from oxidation of crude anilines 
showed that mauve actually was obtained because of the presence of impurities (tolui- 
dines) in the starting material. Mauve and the other dyes of the azine and complex 
azine group are derivatives of the chromophore phenazine. The research momentum 
involving aromatic amines continued with DeLaire, et al. (1867), preparing diphenyl- 
amine by heating aniline in the presence of aniline hydrochloride. The first thiazine 
dye, Lauth's violet, was prepared by Lauth in 1876 by the ferric chloride oxidation of 
phenylenediamine in the presence of hydrogen sulfide. Caro applied Lauth's reaction 
to N, N -dimethyl -p-phenylenediamine and obtained the industrially important dye 
jxiethylene blue. It was at this stage that Bernsthen, a research chemist in Heidelberg, 
investigated the structures of Lauth's violet and methylene blue. Bernsthen reasoned 
that unequivocal proof of structures could best be attained by independent synthesis. 
This concept led him to the synthesis of phenothiazine by heating diphenylamine in the 
presence of sulfur. He then showed that Lauth's violet could be derived from phenothi- 
azine by nitration, reduction, and oxidation; and methylene blue by nitration, reduc- 
tion, alkylation, and oxidation. By the time Bernsthen's proof-of-structure studies and 
synthesis of phenothiazine were published in 1883, he had secured German patent rights 
on the latter. Bernsthen's synthesis of phenothiazine and proof of structures of Lauth's 
violet and methylene blue were based on a continuous feedback between a basic science 
and its industrial application. This approach facilitated and accelerated the develop- 
ment of both the dye industry and organic chemical research. The synthesis, identifi- 
cation, and ready availability of phenothiazine and its derivatives pointed the way to 
new biological and industrial applications. 

The greatest initial impact of the synthesis of phenothiazine was on the dye in- 
dustry. Favorable world markets for dyes, the commercial availability of raw mate- 
terials for the preparation of dyes (coal tar, chemicals, nitric acid, sulfur, sulfuric 



G-3 

acid iron, hydrochloric acid) and adequate technical skills in both the industrial and 
academic settings catalyzed the immediate development of many phenothiazine dyes. 



Search for Synthetic Antihistaminic Drugs 



The emergence of phenothiazine derivatives as drugs for psychoses was closely 
associated with the development of a group of phenothiazines that have antihistaminic 
properties. The development of synthetic antihistamines during the early part of the 
20th century was facilitated by research which defined the chemical, pharmacological, 
and clinical properties of histamine and antihistaminic drugs. 

It was long obvious that histamine antagonists would be of great interest, not only 
to those exploring the physiological functions of histamine, but also to physicians con- 
cerned with the treatment of allergenic disorders in which histamine appeared to play 
an important role. However, a quarter of a century elapsed between the description 
of the remarkable pharmacological actions of histamine by Dale and Laidlaw (I9IO), 
particularly in relation to analphylactic shock, and the discovery of drugs with signifi- 
cant antihistaminic properties. Antihistaminic activity was first detected by Bovet and 
Staub in 1937 in one of a series of phenolic ether amines synthesized by Fourneau in 
1910. This compound, 2-isopropyl-5-methyl phenoxyethyldiethylamine, protected 
guinea pigs against several lethal doses of histamine, antagonized histamine -induced 
spasms of smooth muscles, and most significantly, lessened the symptoms of an- 
alphylactic shock. Although the drug was too ineffective and too toxic for clinical use, 
it provided evidence from the study of congeners that toxicity and antihistaminic activity 
did not run parallel. Thus, in 1939, Staub reported that diethylaminoethyl-N-ethylaniline 
was a stronger histamine antagonist than the first compound tested. Unfortunately, the 
second compound still was too toxic, but a dimethylamine derivative, phenbenzamine, 
investigated by Halpern in 1942, proved acceptable for clinical use. Phenbenzamine, 
marketed by Rhone -Poulenc as Antergan, became the first antihistamine to be used 
therapeutically. However, therapeutic use of phenbenzamine revealed that the drug had 
an unexpected effect on the central nervous system (CNS) in man — one that had not been 
seen in laboratory tests with guinea pigs, rats, and mice. In these animals, Halpern 
(I942) observed that large doses of phenbenzamine usually produced "psychic excitation", 
while in humans the antihistamine seemed to have a sedative effect. 

At this stage, Rhone-Poulenc decided to strengthen its position in drugs. Besides 
being used as an insecticide (Campbell, et al. , 1934) and an anthelmintic agent 
(Harwood, et al. , 1938), the use of phenothiazine derivatives as antimalarials was sug- 
gested independently by researchers in France and in the United States in 1944. Their 
suggestions were based upon the already established antimalarial properties of meth- 
ylene blue and methylene blue derivatives (Guttman and Ehrlich, 1891; Fourneau, et al. , 
193 I; Schuleman, 1932). Accordingly, Oilman and Shirley in the United States in 1944 
prepared a series of nonoxidized phenothiazine derivatives which contained a basic 
alkylaminoalkyl group attached to the nitrogen of phenothiazine. None of these com- 
pounds showed significant antimalarial activity. In retrospect, had the phenothiazine 
derivatives prepared by Oilman and Shirley been subjected to a systematic pharmaco- 
logical screening, the route leading to CPZ naight have been discovered in the United 
States. In an appraisal of the antimalarial data, the investigators suggested the exam- 
ination of some nonoxidized phenothiazine derivatives containing, in addition to the basic 
side chain, two nuclear substituents drawn from groups like methyl, methoxy, chloro, 
or diethylamino. As discussed below, Charpentier independently used part of this 
rationale in his investigations leading to the preparation of CPZ. Owing to disruptions 



G-4 

in communication caused by World War II, Charpentier and his associates at Rhone- 
Poulenc were unaware of the results of the American studies. At the time, this lack of 
communication seemed to be unfortunate because the French investigators reached the 
same conclusions from their work as did Oilman, et al. , but they did not stop with ex- 
amination of the compounds on avian malaria. Instead, a systematic study of the phar- 
macological properties by Halpern, et al. (1946) of the new phenothiazine amine deriva- 
tives that Charpentier and his colleagues had prepared showed that one of them, 
fenethazine, had strong and long-lasting antihistaminic properties. This encouraged 
Charpentier to synthesize other phenothiazine amine derivatives structurally similar to 
fenethazine. Of the first seven compounds tested, promethazine surpassed even 
fenethazine in antihistaminic activity, and like many antihistaminic drugs, showed a 
strong sedative effect. 

Rhone -Poulenc 's interest in the phenothiazine amines was furthered by Sigwald's 
observation (1946) that ethopropazine is effective against the tremor and rigidity of 
parkinsonism. Sigwald's investigation verified Bovet's observation (1946) on the effects 
of ethopropazine on sympathetic and parasympathetic nerve functions in dogs and 
rabbits. 

Meanwhile in October, 1947, Ducrot demonstrated promethazine's sedative effect 
in causing a marked prolongation of barbital-induced sleep in the rat and the rabbit. 
Additional sleep potentiation studies by Bovet on phenothiazine amines and antihista- 
mines synthesized by Rhone -Poulenc indicated that promethazine far surpassed them 
in its sedative or hypnotic properties. 

The establishment of reliable tests to determine the therapeutic index of a drug 
with CNS activity (such as had been observed by Ducrot) was a key research need at 
this time. To eliminate this void, two tests were developed. Winter (1947) developed 
a test to measure the antihistaminic potentiation of the sedative action of barbiturates. 
In 1948, Macht, et al. , reported on the use of conditioned reflex tests (rope climbing) 
in laboratory animals to identify a CNS effect of synthetic antihistamines. In 1949, 
Winter and Flataker, using Macht's rope-climbing test, reported that synthetic anti- 
histamines, promethazine, and ethopropazine significantly reduced the climbing time 
of rats by amounts that varied with the compound and the dosage. 

The tests by Winter, Winter and Flataker, and Macht provided a means for 
routinely screening new compounds to detect and estimate their sedative effects on 
man. At the same time, they provided researchers with tecliniques needed to explore 
and characterize CNS effects of drugs. The data by Winter and Flataker on the CNS 
effects of promethazine was an important factor in Rhone-Poulenc 's decision to develop 
a phenothiazine amine with a strong CNS effect. The two tests, barbiturate potentia- 
tion and conditioned reflex, were the principal methods used to assay the phenothiazine 
derivatives for CNS effects. 

In 1950, Laborit introduced promethazine into clinical anesthesia as a potentiating 
agent. His recognition of the therapeutic value of promethazine's secondary properties 
had led him to expand the use of synthetic antihistamines (1) as preanesthetic agents 
and for postoperative management and (2) to potentiate general anesthetics, thereby 
reducing the amount of general anesthesia required for narcosis and thus minimizing 
the possibility of shock. 



G-5 
Synthesis and Evaluation of Chlorpromazine 



In 1950, the need for other phenothiazine derivatives with potentiating actions as 
well as greater CNS activity was given first priority by Rhone -Poulenc. Accordingly, 
in October, 1950, Dr. Pierre Koetschet, Assistant Scientific Director at Rhone- 
Poulenc, drafted a research proposal explaining why he felt Rhone-Poulenc should 
attempt to develop a phenothiazine derivative with maximally active CNS effects, out- 
lined a research plan, and discussed the compound's potential application in general 
anesthesia, drug antagonism, antiparkinsonian therapy, psychiatry, and epilepsy. 

Koetschet's proposal was accepted quickly and within a week the plan was put into 
action. In retrospect, a careful appraisal by Koetschet of the chemical, biological, 
pharmacological, and medical developments up to 1950 was a decisive factor that led to 
his research inemorandum. Laborit's work on the use of phenothiazine amines in 
surgery, especially as a potentiator, 'and a timely report by Carlisle and Crescitelli 
(I95O) concerning the miode of action of antihistamines on the nervous system also were 
strong factors in the formulation of Koetschet's research proposal. 

In the research leading to the discovery of chlorpromazine at Rhone-Poulenc 's 
laboratories, Charpentier was responsible for the cheraical work and Madame S. 
Courvoisier was in charge of the pharmacologic assays, using tests developed earlier 
by Ducrot, Macht, and Winter. The research purpose now was to discover compounds 
that had central nervous system activity rather than antihistaminic effects. When 
Courvoisier found that a compound closely related to promethazine had a far stronger 
local analgesic action than histaminic effects, the research emphasis shifted to 
Charpentier 's laboratory. Charpentier had just prepared a new compound, 2- 
chlorophenothiazine, by heating 3 -chlorodiphenylamine in the presence of sulfur. 
(This was an adaptation of Bernsthen's 1883 synthesis of phenothiazine. ) The methyla- 
tion of 3 -chlorodiphenylamine with sodium followed by condensation of the sodium de- 
rivative with 3-dimethylamiinopropylchloride yielded CPZ. Courvoisier 's pharmaco- 
logical evaluation of CPZ, which included using a modified version of the Macht- 
Winter rat rope-climbing test, showed that the compound had exceptional CNS effects. 
By mid-March, 1951, Rhone-Poulenc felt that the pharmacological data justified clin- 
ical trials of chlorpromazine. Thus, in approximately 5 nnonths, CPZ went from 
synthesis to pharmacological assays to clinical trials. As discussed below, CPZ 
opened a new frontier in psychotherapy; its earliest clinical use, however, was in 
anesthesiology. 

Early Clinical Psychiatric Trials With 
Chlorpromazine 

On April 6, 1951, Therapeutic Research Center of Specia (pharmaceutical divi- 
sion of Rhone-Poulenc) received the first shipment of CPZ to distribute for clinical 
pharmacological tests by physicians residing primarily in the Paris area. From 
September 9, 1951, to March 10, 1952, Rhone-Poulenc shipped 3,492 samples to 118 
clinical investigators in France and 8 foreign countries (England, Switzerland, Algeria, 
Argentina, Senegal, Belgium, Italy, and The Netherlands). 

Data from three of the clinical investigations in these early trials gave Rhone- 
Poulenc a strong impetus to develop CPZ's uses in psychiatry: (1) Schneider's studies 
in 1951 on barbiturate potentiation with chlorpromazine in a patient with severe manic 
agitation and two patients undergoing narcoanalysis; (2) observations of CNS effects by 



G-6 

Laborit, et al. (1952) during the confirmation of four principal uses for CPZ, (a) as an 
anesthetic potentiator, (b) for preventive management of surgicaL shock and postopera- 
tive complications, (c) for artificial hibernation, and (d) for the treatment of shock; 
(3) Sigwald's use of diethazine and other phenothiazine amine derivatives in 1946 to 
alleviate the symptoms of Parkinson's disease. Although Sigwald and Bouttier (1951) 
found CPZ to be ineffective in treating Parkinson's disease, they found it effective in 
treating nonhospitalized ambulatory patients suffering from psychotic disorders. 

The above observations led to investigations of treatment modalities. Hamon 
(1952) concluded that barbiturate potentiation with CPZ rapidly ameliorated anxiety 
states of hospitalized patients and envisaged possible use of the potentiator for out- 
patient management. Abely (1952) observed the utility of CPZ in shock therapy for the 
treatment of anxiety states and manic depressive states. 

Deschamps (1952) found that CPZ was an important new drug for sleep therapy 
involving agitated patients. Gachkel and Brisset (1952) confirmed Deschamps' conclu- 
sion, but Ey and Berard (1952) obtained inconclusive results in their sleep-therapy 
expe riments . 

The most significant form of CPZ therapy, its administration alone, was investi- 
gated by Sigwald and Bouttier in 1951-52 at St. Anne's Psychiatric Center in Paris. 
They presented the results of their work in 1952 in three papers on CPZ therapy to the 
members of the Societe Medico-Psychologique and in three other papers in 1952 to the 
50th French Congress of Psychiatry and Neurology. The detailed clinical research on 
chlorpromazine triggered the eventual international revolution in the treatment of 
mental disease. The work by Sigwald and Bouttier, combined with the studies of 
Laborit and Huguenard on the potentiating action of CPZ and Deschamps' observations 
on the activity of CPZ on psychotic patients, had the greatest influence on Rhone- 
Poulenc to emphasize the use of CPZ in psychiatry. 

The period 1952-1954 marked the dissemination of CPZ in countries outside of 
France. It was marketed in England and Italy under the trade name Largactil (a reflec- 
tion of the large nuraber of actions manifested by CPZ as described by Courvoisier in 
1953). These included gangliolytic, adrenolytic, antifibrillatory, antiedemic, antipy- 
retic, antishock, anticonvulsant, and potentiator actions of a number of analgesics and 
central depressant drugs. 

CPZ was marketed in Germany as Megaphen. A summary of the dissemination 
of CPZ and technical publications and technical meetings resulting from research dur- 
ing this period on the disseminated CPZ is given in Table G-1. 

The results of some of these early investigations strongly supported CPZ's role 
in psychiatry. In June, 1953, Staehlin and Kielholz published the first Basel Clinic 
paper on CPZ in psychiatry. Their work confirmed the earlier observations by Delay 
and Deniker. This initial success led to the administration of CPZ to over 200 of the 
500 Basel Clinic patients experiencing psychotic disturbances. In 1954, Elkes and 
Elkes conducted a 22-week study on 12 male and 15 female psychiatric patients in which 
the patients served as their own controls by being given CPZ and a placebo alternately. 
These investigators concluded that the patients became more manageable during the 
medication period. The growing medical importance of CPZ led in November, 1953, 
to the Basel Clinic symposium on Largactil, the first conference to deal solely with 
CPZ therapy in psychiatry. 



G-7 



TABLE G-1. DISSEMINATION OF CPZ AND TECHNICAL 
PUBLICATIONS AND MEETING RESULTING 
FROM RESEARCH^^* 



Shipments by Rhone-Poalenc for Clinical Trial Outside 
France, September 195 1 -February 195Z 

9/22/51 ZO samples to May and Baker, Ltd., England 

10/8/51 5 samples to Professor Jentzer, Switzerland 

10/30/51 5 samples to Dr. Fanjeaux, Algeria 

11/7/51 10 samples to Dr. Bilfeld, Argentina 

11/7/51 5 samples to Dr. Letac, Indonesia 

12/20/51 2 samples to Dr. Leroy, Belgium. 

1/9/52 5 samples to Professor Agostino, Trapani, Italy 

1/15/52 5 samples to Professor Jentzer, Switzerland 

1/22/52 5 samples to Dr. Crul, The Netherlands 

1/22/52 5 samples to Dr. Monjal, Algeria 

1/22/52 5 sainples to Dr. Goldblat, Belgium 

2/4/52 2 samples to Dr. Saman, Belgium 

2/12/52 4 samples to Ets Barberot, Switzerland 

Dissemination and Publications, 1952-1954 

4/52 Paper by S. Rigotti, Padua, Italy - first non-French 

publication on CPZ in psychiatry 
5/52 First samples sent from Rhone -Poulenc to Smith, Kline & 

French in Philadelphia 
8/52 First German clinical trial with CPZ - use in surgery and 

for potentiated anesthesia and artificial hibernation, by 

Irmer and Koss in Dusseldorf 
11/29/52 Paper by Arnold, Hift, and Solms, Vienna, on CPZ in 

psychiatry 
1/53 Labhardt brings CPZ to Basel from Delay's clinic in Paris 

3/53 Paper by Irmer and Koss on CPZ for surgery - first 

German clinical publication 
Spring 1953 Reports on CPZ's use by French military surgeons in 

Indochina (Nicol 1953) 
6/53 Paper by Staehlin and Kielholz, Basel Clinic - first Swiss 

publication on CPZ in psychiatry 
Spring 1953 ■ Heinz Lehmann, Montreal, receives first samples of CPZ 

10/53 Paper on CPZ as antiemetic drug by Friend and Cummins, 

Boston, Mass. — first U. S. publication on CPZ 
11/53 "Largactil Symposium" held at University of Basel - first 

symposium on CPZ in psychiatry 
12/53 Paper by D. Anton-Stephens received for publication — 

first British report on CPZ in psychiatry 
2/54 Paper by Lehmann and Hanrahan — first North American 

publication on CPZ in psychiatry 
4/54 Anton-Stephens' paper published in Journal of Mental 

Science 
5/54 Paper by N. W. Winkelmann, Jr. , Philadelphia ~ on 

first U. S. work on CPZ in psychiatry 
6/54 Paper by Trelles and Saavedra, Peru — first South 

American publication on CPZ in psychiatry 
9/54 Paper by J. Elkes and C. Elkes, reporting CPZ's first 

evaluation by controlled trial — second British publica- 
tion on CPZ in psychiatry 
9/54 Paper by A. Matarosso, Italy, reporting extensive 

clinical psychiatric study of CPZ 
11/54 Paper by R. R. Webb - first Australian publication on 

CPZ in psychiatry 

(a) Adapted from Swazey (1974). 



G-8 

The first North American publication on CPZ in psychiatry, "Chlorpromazine, 
New Inhibiting Agent for Psychomotor Excitement and Manic States", was authored by 
Lehmann and Hanrahan and appeared in Archives of Neurology and Psychiatry on 
February 4, 1954. This unfunded work was done at Verdun Protestant Hospital (now 
Douglas Hospital) in Montreal. Lehmann and Hanrahan greatly praised CPZ's utility 
in treating manic-depressed patients, and they became convinced CPZ was the answer 
for the syraptomatic control of most cases of severe excitement. For their pilot study, 
they used 71 patients from 18 to 82 years old and gave them unprecedented large doses 
(up to 800 mg/day) of CPZ over a 4-month period. Patient consent was not required. 
Lehmann and Hanrahan felt that CPZ was superior to electroconvulsive therapy and to 
conventional pharmacologic sedation in the management of psychotic disorders. After 
discontinuation of the pilot study, some of the refractory schizophrenics who were in 
the original 71 were left accidentally on large (but unknown) doses of chlorpromazine. 
Three months later, four or five of these chronic backward refractory patients were 
getting better, with chronic paranoids making most progress. This chance experiment/ 
observation, which Lehmann and subsequent investigators confirmed using larger quan- 
tities of CPZ, turned out to be a major contribution to the management of the 
schizophrenic patient. 

In a preliminary study of barbiturate potentiation, Lehmann discovered that in 
normal volunteers CPZ could cause orthostatic hypotension. 

The first report concerning the new drug for the effective treatment of schizo- 
phrenics came from Europe. In 1954, Labhardt published his results on CPZ therapy 
in 205 schizophrenic patients consisting of acute, subacute, and recalcitrant groups. 
Of the total 205 patients, 11 were released after therapy. Consistent with Lehmann's 
observation, 18 percent of the 106 chronic patients in the therapeutically most recal- 
citrant group (ill for more than 5 years) were judged socially cured, 40 percent im- 
proved, and 26 percent slightly improved. Labhardt's observations were confirmed by 
Goldman (1956) at Ohio's Long View State Hospital in Cincinnati, Ohio, in the treating 
of approximately 1, 500 hospitalized patients with CPZ and more than 500 with other 
drugs, chiefly reserpine. Goldman also stressed the effectiveness of CPZ in reducing 
the rate of relapse in patients placed on convalescent status. Of 93 CPZ patients placed 
on convalescent status from August, 1954, to April, 1955, only 6 were returned for 
hospital treatment; of non-chlorpromazine-treatea patients, treated chiefly by electro- 
shock, 59 out of 71 placed on convalescent status returned for additional hospital 
therapy. 



Chlorpromazine in the United States 

CPZ's migration into the United States began in April, 1952, when J. Monet, 
Rhone-Poulenc 's Director for Agreements and Patents, wrote a letter to Francis Boyer, 
President of Smith, Kline and French Laboratories (SK&F). Monet enclosed several 
documents supportive of CPZ's potential in mental disease and also asked if SK&F 
would be interested in a licensing agreement with Rhone-Poulenc to market CPZ in the 
United States . 

Two years later, in May, 1954, after SK&F had substantiated much of the earlier 
pharmacological and clinical data supportive of CPZ's potential and had addressed 
themselves to assessment of its side effects, SK&F began to market CPZ as Thorazine. 
Within 8 months, CPZ was given to approximately 2 million patients. SK&F's sales 



G-9 

volume increased by approximately one-third within a year of Thorazine's introduction. 
Much of SK&F's fiscal growth from net sales of $53 million in 1953 to $347 million in 
1970 can be attributed to Thorazine. 

The acquisition of CPZ by SK&F represents a major success in the company's new 
research orientation. When Boyer became Executive Vice President in charge of re- 
search in 1936, he and SK&F's President, C. M. Kline, decided that SK&F should con- 
centrate on developing, manufacturing, and marketing trademark products advertised 
only to the medical profession. To maintain a flow of trademark products in this enter- 
prise, they felt it would require a close working relationship on a national and inter- 
national basis between a pharmaceutical company, academic research, scientists, and 
clinicians. It was in such a management and research atmosphere that SK&F acquired 
CPZ from Rhone -Poulenc and Rhone-Poulenc received in exchange SK&F 525, a liver 
enzyme detoxicant. 

Major events at SK&F Laboratories leading to the marketing of Thorazine, brand 
name for SK&F's chlorpromazine, are given in Table G-2. Throughout these develop- 
mental studies, SK&F members of the Thorazine team maintained a feedback of infor- 
mation in their laboratory, sought and obtained exchange of valuable pharmacological 
and clinical data from Rhone-Poulenc personnel, visited in Europe, and had chlorpro- 
mazine investigators from Europe visit SK&F. In this respect, Laborit's visit to the 
United States in 1953 to deinonstrate chlorpromazine 's potential in artificial hiberna- 
tion gave such discouraging results that interest in chlorpromazine for this purpose 
was lost. This realization greatly accelerated SK&F's effort in setting research pri- 
orities on Thorazine's many other clinical applications, obtaining the necessary data to 
gain clinical acceptance and FDA approval, and finally establishing a viable marketing 
program. 

Only a few figures are available concerning the cost of the development of Thora- 
zine. Marketing of the drug was delayed from January until May, 1954, during which 
period an intensive clinical study was initiated to determine Thorazine's implication in 
liver dysfunction. After 4 years (1953 to 1957) and a cost of about $1 million, no defini- 
tive answers had been obtained. In April, 1953, the Thorazine Project Team at SK&F 
projected expenditures of $200, 000 in development of clinical psychiatric data between 
January, 1953, and April, 1954, besides an estimated $50, 000 in research funds spent 
in 1952. Through Jione, 1954, the Development (clinical investigation) Committee of 
SK&F estimated that grants for clinical work would total $122,600, while the grants for 
clinical psychiatry through June, 1953, would total only about $11, 000. 

Shortly after CPZ was cleared for marketing, Boyer, on a nationally televised 
program produced by SK&F, stated that SK&F research people had spent up to that time 
about 60, 000 man-hours on Thorazine. 

The first U. S. clinical investigative report on Thorazine, "New Antiemetic Drug: 
Preliminary Report", by Dr. D. Friend and Dr. J. F. Cummins of Peter Bent Brigham 
Hospital was published in JAMA , October 3, 1953. The first U. S. publication ( JAMA , 
May 1, 1954) on chlorpromazine in psychiatry entitled "Chlorpromazine in the Treat- 
ment of Neuropsychiatric Disorders" was authored by Dr. William Winkelman, Jr., of 
Philadelphia's Sidney Hillman Medical Center. Winkelman's pilot study consisted of 
142 psychoneurotic patients who received CPZ for 2 to 8 months. The results of his 
study were supportive of CPZ's utility to reduce severe anxiety, quiet manic or ex- 
tremely agitated patients, and change the hostile, mentally disturbed elderly patient into 
a manageable patient. His data also reflected the drug's usefulness in treating ambula- 
tory, noninstitutionalized patients. 



G-10 



TABLE G-2. MAJOR EVENTS IN U. S. LEADING TO THE 
MARKETING OF THORAZINE(a) 



Year 



Month 



Event 



1952 April 



May- 



July 
August 

September 
October 

December 

1953 January 

March 

May 

June 

July 

July- 
December 

September 

October 

November 

December 

1954 March 
March 



May 



SK&F offered CPZ by R-P. Review French data and decide to 
seek U. S. rights. Boyer & Bo reach verbal agreement on 
CPZ and SK&F 525. 

SK&F receives first 200 g of CPZ from R-P after preliminary 
licensing agreement signed. Animal pharmacologic tests be- 
gin. Scull urges virork on CPZ's neurologic and psychiatric 
uses. 

CPZ should be ready for human trials by September 1 

Subacute toxicity tests in rats and dogs uniformly negative. 

CPZ safe for preliminary clinical trials. Begin plans for four 
clinical trial areas. 

Research group submits 25-item pharmacology report. 

First clinical tests begin: nausea and vomiting, mania, itching, 
hypothermia. Tested for dosage, safety, effects on 63 patients 
through December 16, 1952. 

Patent office rejects R-P's patent application for CPZ. 

CPZ transferred from Research to Development. Four areas 
recommended for full clinical trials: nausea and vomiting, 
sedation-general medicine, sedation-psychiatry, antipruritic. 

Winkelman begins clinical psychiatric trial. 

SK&F begins o-wn manufacturing of CPZ. 

R-P's patent application granted. 

SK&F formally tells R-P that it will market CPZ in the U. S. 

Side effects crises — jaundice, hypotension, dermatitis. 

Thorazine selected as brand name for SK&F CPZ. 

Friend and Cummins; first U. S. paper on CPZ (as antiemetic). 

Development Committee begins formulating plans for national 
introduction of Thorazine. 

SK&F personnelrneet with FDA on submission of New Drug 
Application for Thorazine. CPZ tested on 104 psychiatric 
patients and on 1000+ patients as antiemetic. 

New Drug Application submitted to FDA. 

FDA approves Thorazine for nausea and vomiting and 
neuropsychiatry. 

Thorazine marketed, 26 months after compound CPZ offered 
to SK&F. 



(a) adapted from Swazey ( 1974). 



G-U 

The psychiatric investigations in 1954 by V. Kinross -Wright, Baylor University 
College of Medicine, confirmed Lehmann's earlier observations that CPZ had not only a 
sedative effect on patients but made them less refractory to psychotherapy. The re- 
sults of the clinical trials by Winkelman, Lehmann and Hanrahan, and Kinross -Wright 
influenced strongly SK&F's decision to market Thorazine for neuropsychiatric use. 

The importance of the results of the clinical trials by the above investigators 
needs also to be expressed in terms of American psychiatry in the middle of the 20th 
century. American psychiatry was dominated by the psychotherapists and somato- 
therapists. To their reluctance to accept a novel psychiatric drug can be added the 
skepticism with which American psychiatrists viewed the results and practices of 
their European counterparts. These conditions are reflected in the observation that 
CPZ had been tested in only 104 psychiatric patients by the end of 1953, while its 
effectiveness had been clearly established in 1, 000 patients as an antiemetic drug. 
Clinical data obtained by Lehmann and Hanrahan, Winkelman, and Kinross -Wright 
helped to fracture the ideologic barrier and rapidly advance the clinical applications of 
CPZ. 

The beneficial effects of CPZ in psychotic and neurotic disturbances and the revo- 
lutionary changes it helped to initiate in the operation of hospitals and management of 
mental patients might be viewed as sufficient stimulus to accelerate clinical trials and 
acceptances. This was not the case, since several sources of opposition developed and 
several educational and financial gaps were recognized. In addition to opposition to 
usage of CPZ in office-practice psychiatry (discussed above), many institutional bud- 
gets were inadequate or nonexistent for CPZ use, hospital personnel lacked knowledge 
and training concerning chlorpromazine 's therapeutic values, and civic community and 
legislative organizations were unfamiliar with treatment costs and custodial-care costs. 
Especially damaging to SK&F's efforts was the feedback from California about the large 
number of released patients returning to the mental hospitals. SK&F discovered that 
the reason was that the family physician put the discharged patient on a low mainte- 
nance dose of Thorazine which did not ameliorate his behavioral disorders. 

The problems listed above that arose in the dissemination and in the psychiatric 
usage of Thorazine were resolved by SK&F's Task Force. This team of 50 men was 
organized in 1954 and from 1955-1960 became known as the Hospital Sales Service. 
One member of the force was assigned to each state capital to assist community groups 
and the legislature in developing the necessary budgets for state mental hospitals. The 
Task Force also was involved in the dissemination of information concerning Thora- 
zine's side effects, working with officials of the Veteran's Administration in Washington, 
D. C. , to evaluate their drug programs, organizing and sponsoring symposia dealing 
with the discharged patient and his new world, working with and funding rehabilitation 
programs in mental hospitals, and developing a remotivation program in cooperation 
with the American Psychiatric Association. 

The years 1955 and 1956 generated national and international conferences and pub- 
lications concerning the new psychiatric weapons. These events are summarized below. 

April 1955 Barcelona, Spain First major conference on CPZ 

June 1955 Philadelphia, Pa. Symposium on "CPZ and Mental Health" 

September 1955 Italy Conference on CPZ 

October 1955 Italy Conference on CPZ and reserpine 



G-12 

October 1955 Paris, France First International Colloquium on CPZ 

and Neuroleptic Drugs in Psychiatric 
Therapy 

1955 Rhone-Poulenc Issuance of bibliography on CPZ 

19*^5 International Record of Published "Symposium on CPZ in 

Medicine (U. S. ) Clinical Medicine " 

1956 Journal of Clinical and Published "International Symposium on 

Experimental Psycho- CPZ" 

pathology; International 
Record of Medicine 

1956 Psychiatry and Neurology Research conference on chemotherapy 

Service, U. S. Veterans in psychiatry 
Administration 

1956 Interim Committee, Treatment of mental illness 

California Senate 

In 1955 and 1956, a national survey by the California Senate's Interim Committee 
on the Treatment of Mental Illness to determine the use made of the new antipsychotic 
drugs in other state hospitals indicated that about two-thirds of the states were finan- 
cially supportive of the new approach to the treatment of psychiatric disorders. 

During the mid-1950's, not all the activity in mental research was focused on 
CPZ, Instead, expansion of the pattern of multidisciplinary research and clinical tests 
that assured CPZ's entrance and acceptance in psychiatry led to (1) the reexamination 
of available drugs and the synthesis of many new products for the treatment of mental 
disorders and (2) the development of improved methodology for the pharmacological and 
clinical evaluation of drugs. The societal impact of these developments was the sudden 
availability of a wide selection of antianxiety and antipsychotic drugs. For example, 

(1) The antianxiety agents, specifically the subclass with CNS effects, 
were a product of this period. 

(2) The introduction of the first two antidepressants was strongly in- 
fluenced by the technological diversification that gave us CPZ. 
Iproniazid, the first of the MAO inhibitors, was synthesized in 1951 
at the Hoffmann-LaRoche Laboratories as an antituberculotic agent. 
However, adverse side effects, including euphoria, precluded its use 
in the treatment of tuberculosis. Recognizing this side effect led 
Kline and Loomer in 1957 to develop iproniazid as an antidepressant. 
Earlier, Kline had done research on reserpine and CPZ. Imipra- 
mine, which was marketed as an antidepressant by Geigy in 1958, 
first came to the attention of Swiss psychiatrist Kulin who had 
attended the Basel Symposiunn on CPZ. The reports he heard con- 
cerning the sedative nature of CPZ prompted him to examine imipra- 
mine for possible ataraxic properties and led to his discovery of 
imipramine's effectiveness against endogenous depression. 

(3) The growing wave of enthusiasm for use of the rauwolfia alkaloids in 
psychiatry occurred during this period. However, the almost simul- 
taneous appearance of the phenothiazine derivatives, specifically CPZ, 
for treating psychiatric illnesses quickly eroded the strong position of 
the rauwolfia derivatives in psychiatric therapy. 



G-13 

(4) Klerman (1973) summarized the improved methodology that originated 
about this time for evaluating the clinical effects of psychotropic drugs. 
(This methodology also found application in the pharmacological and 
clinical evaluation of many other drugs. ) The psycho-pharmacologists 
greatly improved the double-blind experimental design, expanded the 
use of placebos, devised a number of rating scales, and utilized com- 
putational technology in the therapeutic assessment of drugs. These 
improved methodologies and techniques helped to demonstrate CPZ's 
beneficial impact upon the mental-hospital population (Brill and Patton, 
1957) and firmly established the specific effectiveness of some pheno- 
thiazines, especially CPZ, in acute schizophrenia (NIMH collaborative 
study, 1964). 



STRUCTURE-ACTIVITY RELATIONSHIPS 
OF SELECTED PHENOTHIAZINES 



The potential of phenothiazines in the practice of medicine was indicated in the 
sequence of events that led to the discovery of their therapeutic effects, e.g. , anti- 
psychotic, antiemetic, sedative, hypotensive, and antiparkinsonism effects . An exam- 
ination of the historical development of phenothiazine derivatives suggests that obser- 
vation of effects frequently was based on a single compound. It was not until the 
iinportance of structure -activity relationships in phenothiazine derivatives was investi- 
gated that the chemistry and therapeutic value of these compounds could be properly 
determined. The results of this approach are summarized in Table G-3. 

Chemical substitutions at Jr'ositions 2* and 10* in the phenothiazine molecule play a 
major role in determining drug action. For example, the addition of a substituent at 
Position 2 renders the phenothiazine nucleus asymmetrical and such asymmetry aug- 
ments drug activity. Substitution of a halogen or methoxy group in Position 2 increases 
the potency of phenothiazines for depressing motor activity and conditioned avoidance 
response in animals and for changing psychotic behavior in humans. The substitution 
of a trifluoromethyl group at Position 2 greatly increases the antipsychotic and antie- 
metic potency as well as the tendency to produce extrapyramidal effects. 

The pharmacological activity of the phenothiazine derivatives also is influenced 
by the substituent at Position 10. On the basis of substitution at this site, the pheno- 
thiazines can be divided into four groups. 

(1) The group with an aliphatic side chain, e.g., chlorpromazine, 
promazine, triflupromazine, and methoxypromazine. This is the 
least potent group antipsychotically as can be seen by the dosage 
range. 

(2) The next group with intermediate potency contains a piperidine 
moiety in the side chain and includes mepazine and thioridazine. 

(3) The most potent antipsychotic compounds contain a piperazinyl 
group such as fluphenazine, perphenazine, prochlorperazine, tri- 
fluoperazine, and thiopropazate. They have strong antiemetic prop- 
erties and a greater tendency to produce parkinsonism, but diminished 
sedative effects. They also strongly suppress conditioned avoidance 
responses in animals. 



' In Table G-3, R^ = Position 2 and R-^ = Position 10. 



G-14 



l-H 



X 
H 
O 

W 

Q 
W 

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(4) The phenothiazines used in psychiatry have a 3-carbon bridge between 
the ring and side-chain nitrogen atoms. This is in contrast to anti- 
cholinergic drugs used in the treatment of parkinsonism (e.g. , 
ethopropazide, diethazine) and to antihistaminic phenothiazines (e.g., 
promethazine, isopromethazine). One exception to this generaliza- 
tion of structure and activity is the compound methdilazine vi^hich has 
three carbons between the nitrogen atoms. It produces sedation but 
lacks significant antipsychotic activity. 

Chemical modification of the terminal nitrogen in phenothiazines will alter their 
pharmacological activity. For example, the ability of the phenothiazines to block 
muscarinic actions of acetylcholine is increased by diethyl substitution on the terminal 
nitrogen. Quaternization of the terminal nitrogen diminishes the ability of the result- 
ing compound to pass the blood-brain barrier. 



REFERENCES 

Abely, P., Abdouchell, A., Fourment, J., Schmitz, B., and Delteil, P., Ann. Med. 
Psychol. , 2, 262 (1952). 

Bernsthen, A., Ber. Deutsch. Chem. Ges., _16, 2896 (1883a). 

Bernsthen, A., Ber. Deutsch. Chem. Ges., J_6, 1025 (1883b). 

Bovet, D. , Fournel, J., and Charpentier, P., Therapie, 2, 115 (1947). 

Bovet, D. , and Staub, A. M. , C. R. Soc. Biol., j_24, 547 (1937). 

Brill, H. , and Patton, R. E., Am. J. Psychiat. , 114 , 509 (1959). 

Campbell, F. L. , Sullivan, W. N. , Smith, L. E. , and Haller, H. L. , J. Econ. 
Entom. , 27, 1176 (1934). 

Carlisle, E. M. , and Cresticelli, F. , Science, j_12, 272 (1950). 

Charpentier, P., Gaillot, P., Jacob, P., Gaudechow, J., and Bui-son, P., C. R. 
Acad. Sci., 235 , 59 (1952). 

Courvoisier, S. , Fournel, J. , Ducrot, A. , Kolsky, M. , and Koetschet, P. , Arch. Int. 
Pharmacodyn. , 92, 305 (1953). 

Dale, H. H. , and Laidlaw, P. P., J. Physiol. (London), 4]_, 318 (1910). 

de Laire, G. , and Chapoteaut, C. , Compt. rend., 63, 93 (1867). 

Deschamps, A., Presse. Med., 60, 944 (1952). 

Elkes, J., and E Ikes, C. , Brit. Med. J., 2, 560 (1954). 

Ey, H. , and Berard, E. , Evol. Psychiat., 17, 661 (1952). 



G-16 

Fourneau, E. , J. Pharm. Chim. , 7, 55 (1910). 

Fourneau, E. , Ann. Inst. Pasteur, 46, 514 (1931). 

Gachkel, V., and Brisset, C. , C. R. Cong. Med., Alien. Neurol. France, 50, 491 
(1952). 

Gilman, H. , and Shirley, D. A., J. Amer. Chem. Soc. , 66, 888 (1944). 

Goldman, D. , Chlorpromazine and Mental Health , Lea and Febiger, Philadelphia, Pa. 
(1955). 

Goodman, L. S. , and Gilman, A. , The Pharmacological Basis of Therapeutics , 
4th Ed. , 855, The Macmillan Company (1970). 

Guttman, P., and Ehrlich, P., Klin. Wochenschr. , 28, 953 (1891). 

Halpern, B. N. , Arch. Int. Pharmacodyn. Ther. , 68, 339 (1942). 

Halpern, B. N., and Due rot, R., C. R. Soc. Biol., 140 , 361 (1946). 

Hamon, J., Paraise, J., and Velluz, J., Ann. Med. Psychol., 110 , 331 (1942). 

Harwood, P. D. , Sci. Mo., 62, 32 (1946). 

Ihde, A. J. , The Development of Modern Chemistry, Harper and Row, New York; ref. 
Cannizzaro, S. , 226, and Kekule, F. , 310 (1964). 

Kinross -Wright, V., Postgrad. Med., j_6, 297 (1954). 

Kirk, R. E. , and Othmer, D. F. , Encyclopedia of Chemical Technology, 1, 914, 
Interscience Encyclopedia, Inc., New York (1947); contains aniline chemistry refer- 
ences: Unverborden, 1826; Runge, 1834; Fritzsche, 1840; Zinin, 1842; Hoffmann, 
1843; and Perkin, 1856. 

Kirk, R. E. , and Othmer, D. F. , Encyclopedia of Chemical Technology , 2, 421, 
Interscience Encyclopedia, Inc., New York (1948); contains benzene chemistry refer- 
ences: Faraday, 1825; Mitscherlich, 1833; Hoffmann, 1845; and Mansfield, 1848. 

Klerman, G. L. , Drug Therapy , 3, 28 (1973). 

Koetschet, P., Int. Rec. Med., 168 , 295 (1955). 

Labhart, F. , Presse. Med., 56, 170 (1954). 

Laborit, H. , Acta Chir. Belg. , 48, 485 (1949). 

Laborit, H. , Presse. Med., 58, 138 (1950). 

Laborit, H. , Presse. Med., 58, 416 (1950). 

Laborit, H. , Anesth. Anag. Reamin. , 1_, 290 (1950). 

Laborit, H. , Sem. Hop. Paris, 26, 3646 (1950). 



G-17 

Laborit, H. , Ann. Med. Psychol., n_0, 403 (1952). 

Lauth, C. , Ber. Deutsch. Chem. Ges., 9, 1035 (1876). 

Lehman, H. E. , and Hanrahan, G. E. , AMA Arch. Neurol. Psychiat. , 7j_> 227 (1954). 

Loomes, H. P., Saunder, J. C., and Kline, N. S. , Psychiat. Res. Rep., Amer. 
Psychiat. Assoc, 8, 129 (1957). 

Macht, D. I., and Hoff master, T. , Fed. Proc. , 7, 242 (1948). 

National Institute of Mental Health, Psychopharmacology Service Center Collaborative 
Study Group, Arch. Gen. Psychiat., J^, 246 (1964). 

Schulemann, W. , Proc. Roy. Soc. Med., 25, 897 (1932). 

Sigwald, J., and Bouttier, D. , Ann. Med., 54, 150 (1953). 

Sigwald, J., Bovet, D. , and Dumont, G. , Rev. Neurol., T^, 581 (1946). 

Staehlin, J. E. , and Kielholz, P., Schweiz. Med. Wocherschr . , 83, 581 (1953). 

Staub, A. M. , Ann. Inst. Pasteur, 63, 400 and 485 (1939). 

Swazey, J. P. , Chlorpromazine in Psychiatry: A Study of Therapeutic Innovation, MIT 
Press, Cambridge, Mass., and London, England (1974). 

Winter, C. A., J. Pharmacol. Exp. Ther., 9_0, 224 (1947). 

Winter, C. A., and Flataker, L. , Fed. Proc, 8, 169 (1949). 



SE/ 



S^-i_ 



SEARCH FOR SYNTHETIC ANTIHISTAMINIC DRUGS ANIMAL MODELS FOR CNS ACTIVITY SYNTHESIS AND EVALUATION OF CPZ 



1883 Bemithen Synlhoils □' phonothioiino — 
MR/D/W/M,C 

1902 Portlof Oescribod the (vndtome ol 
enaphylactic ihock In dogi 
NR/S/W/P/M 

1910 DBle Observed Ihat Pharmacol ogv ol — 
hitternine rciemblcd syndrome 
of aoBphylactic ihoch 

NR/S/W/P/M 

1910 Fournoau Prepatod thvmonyeihvldioihyl- 
amine (929 F) 

NR/S/W/P/I 



1917 Solloman Shoiwd that hisiamlne causes - 
vosodilsllan ot tho small blood 
vessels and increases peimea- 
bllity ol the capillary walls 
NR/S/U/P/M 

1927 Lewis Observed allergBnic response ol " 
histamine; announced anilgen- 
^ antibodv reaction hypothesis 

NR/S/W/P/M 

1927 Best liolaied histamine as a natural — 

oigaot 

NR/S/W/P/M 

1937 Bouet Oemonstrated antihistamine prop- - 
erlies of thymoxyoihyldiethylamine 
MR/D/W/P/M 

1939 Steub Found diBlhylaminoBihyl-N-eihyl - 
aniline (1571 F) to be a more 
pomerlul antihistamine than 929 F 
MR/D/W/P/M 

1939 Rhone-Poulenc Laboiaioties Launched 
large-wale program on synthesis 



MR/D/W/P/C 



1942 Halpern Found that phenbenz 

10 times mors powerful than 
1571 F and considerably less 
toxic; noted sedative effects in 
humans and "psychic excitation" 

MR/S/W/P/C 

1942 Rhone-Poulenc Laboratories Phentxnzamino 
marketed as Antergan. became first 
antihistamine to be used iherapeu- 



1942 Rhone-Poulone Latwratoriei Developed 

1B45 Neoanieigan, Benadryl, and 

Pyribonzamine; sedation most 
consistent side effect of H-P's 
synthetic antlhittamlnlcs 
MR/S/W/P/C 

1945 Halparn Found antihlsiaminic properties — 
in compounds prepared by 
Charpentiar in 1944 
MR/S/W/P/C 

1945 Rhone-Poulenc Laboratories Phanolhiazine — 
chemistry mergad with research on 
synOietic antihistamlnics 
MR/D/W/P/C 

1946 Charpentier Prepered fanelhazine — strong — 
end long-lasting antlhlstaminlc 
properties 

MR/D/W/P/C 

1947 Charpeniief Prepared promothaiina 

(Phenerganl; encollent onti- 
hittamlnic: sedative elfoci 
observed by Halparn 11949) 
MR/S/W/P/C 

1946 Sigwold Reported on antiparkinsonian ■^— 

1947 oflecis of diothailne; also 
observed sedative ellect 

MR/S/W/P/M.C 

1947 Laborit Concluded ihot chemical media- — 
tors ploy key role in shock 
MR/S/W/P/M 

1949 Laborlt In an S-month cllnicol ttinl. lor " 
managing shock and other post- 
properties of phonothiailne omine- 
danved anithisiamioos eliminated 
the need ot morphine 

MR/S/W/P/M,GL 



1950 Laborli Phonolhlazino a 

histamines found valuable os pre- 
onefthellc agents, postoperative 
monagemani, end potentiator ol 
general anesthetics 

MR/D/W/P/M,GL 



1943 Macht Rat rope climbing test 

MR/D/U/G/M 

1947 Ducrot Developed lest to detect CNS 
Macht actions of antihistamlnics in 
Winter animals — barbiturate potenda- 



MR/D/U,W/G,P/M,C 



1950 Koetschet Outlined research plan thai 
led to synthesis of CPZ 
MR/D/W/P/C 

1950 Courvoisier Pharmacological tests ol 
some compounds closely 
related to CPZ demon- 
Ill powerful antlhiftamine 
propartiH, 



nalgesic 



(21 strong ai 
properties 
MR/S/W/P/C 



CPZ IN OTHER COUNTRIES 

1952. April Rigotie. Italy First non-French pub- 

lication on CPZ in 
psychiatry 

1953, January Labhardt Brought CPZ to Basel 



1953, November Largactll First lymposlom on 

Symposium CPE In ptychialry. 
University of Basel 

1964, April An ton -Step hens First British publi- 
cation on CPZ in 
piychletry 

1954, htoy Winkelman First North American pub- 

licailon on CPZ in 

1964, June Tralles (Peru! First South American 
publication on CPZ in 
psychiatry 



1951 Charpentier Addressing himself to strong 
analgesic properties, decided 
to add 3-dimethylBmino- 
propyl group to the nitrogen 
atom ol 3-chlorophenothla- 
zine (a compound he had 
just prepered): obtained 
CPZ 12/11/50 

MR/D/W/P/C 

■ 1951 Courvoisier CPZ found to possess a wide 
range of desirable pharma- 
cologic properties including: 
1-Low toxicity 
2-G8nglloplegic properties 
3-Sympatholytic properties 
4-Weakly spasmolyllc 
5-Weakly vasodilatory 
6-Weak analeptic activity 
7-Weak antihistaminic 

8-A nest hat ic potentiator 
9-Antithermal and hypo- 

ihermal activity 
10-Strong aiHiematic activity 
ll-Promotes psychic disorlen- 

12-Local anesthetic 
13-Active in hemorrhagic and 

14-RBnal function unimpaired 
MR/D/W/P/C 



19S1 Therapeutic Received first samples 

Research Center of CPZ for clinical 
of Specia testing 

MR/D/W/P/C 

■ 1951 Schneider Observed barbiturate poten- 
tiation with CPZ In manic 
agitation; first direct clinical 
indication ol CPZ's potential 
in psychiatry 

MR/D/W/P/C 



Sigwald Damonstra 



1951- Laborit Confirmed 4 principal uses for 
1952 CPZ: 

1-Anesthotic potentiator 
2-Prevenlive management ol 
surgical shock and post- 
operative complications 
3-Artificial hibernation 
4-Treaiment of shock 
MR/S/W/G/M 

- 1952 Delay Tests at St. Anne's - prolonged 

continuous administration of 
CPZ alone established the standard 
mode of CPZ ihorapy in psychlatrv 
MR/D/W/G/M 

- 1952 Rhone-Poulenc "Note Provlsoire"; pro- 

posed use of CPZ in 
pjychietry - mania, 
schizophrenic detoxlca- 
tlon cures, steep cures 
MR/S/W/P/C 

- 1952 Transmittal of first reports on CPZ - 

Meetings of the Societe 
Medico Psychologique and 
the Luxembourg Congress; 
clinical data published in 
Sociates Annafes, the Con- 
gress Proceedings. La Presso 
Medicale. and L'Evolutlon 
Psychi atria 
MR/S/W/P/C 

- 1962 Rhona-Poulanc Advised tales agents to 

present Largactll (CPZ) 
to three groups — surg- 
eon ■ and Bnesiheiittt, 
neuropsychlatrlsts, and 
obstetricians end gyne- 

MR/S/W/P/C 



CODES 

NR Nonmission-Oriented Research 

MR Mission-Oriented Research 

DV Development 

S Significant Event 

D Decisive Event 

U U.S. Event 

W Foreign Event 

G Government Funding 

P Private Funding 

M Medical School - Hospital - 

University 

I Research Institution 

GL Government Laboratory 

C Company 



CPZ IN THE UNITED STATES 



1952, May Rhone-Poulenc First somple* of 

CPZ sent to SKfiF: 
pharmacological 
studies Initialed 



1953, December SKSF Maiv^thFDAon 

submission ol NDA 
for SK&F'sCPZ 

1954. March 4 SKSF NDA submitted to FOA 

1954, March 26 FOA Approved Thorailno 
neuropfychiatry 

1954. May SKSiF Marketed Thorulne 26 
rtionths alter CPZ offer 

from Rhons-Poulonc 



1954. Novimber Webb Flm Auttrallin publics- 
lion on CPZ in psychiatry 



HISTORIOGRAPH - pHENOTHIAZINES 



H-1 

APPENDIX H. CHLOROTHIAZIDE 

INTRODUCTION AND SUMMARY 



A common pattern in the development of new drugs is not invention de novo but 
rather the exploitation of side effects of existing drugs. An action seen as undesirable 
in one therapeutic context may become the primary drug action in another. 

This case study delineates the development of chlorothiazide and closely related 
compounds as diuretics and antihypertensive agents. Side effects observed initially 
in sulfanilamide led to the discovery of chlorothiazide, a highly effective oral diuretic. 
For their discovery, the scientists shared a $ 10, 000 Albert Lasker Special Award 
(1975). 

Diuresis can be defined as an increase in the rate of urine formation. It may be 
caused by copious fluid intake, by pathological states, or by the action of certain drugs 
(diuretics). Regardless of the cause of urine formation, the site of action of this 
phenomenon is the kidney. The kidney eliminates the waste products of metabolism 
and thereby helps to maintain a constant volume and composition of the body fluids. 
This process is known as homeostasis. Thus, physiologically necessary amounts of 
water, sodium, potassium, and chloride ions are reabsorbed into the blood stream, 
and urea, uric acid, creatinine, water, and excess electrolytes are rejected. It is in 
this manner that the internal environment is maintained. Any marked change in the 
delicate balance between what the kidney excretes and what it retains acts as a warning 
signal indicating that the kidney is not able to fulfill its primary function, which is to 
regulate electrolye concentration and acid-base balance. If this kidney dysfunction 
cannot or is not corrected, it may eventually manifest itself as glomerular nephritis, 
edema, congestive heart failure, and hypertension. Diuretic drugs have been used 
to alleviate the above renal insufficiency. These drugs serve to decrease the reab- 
sorption of water, electrolytes, . and low-molecular- weight organic compounds into the 
blood stream, and as a consequence, to promote the formation of urine. Diuretics 
have been classified into the following groups: 

(1) Osmotic and acidotic 

(2) Xanthines, pyrimidines, and triazines 

(3) Organic mercurials 

(4) Sulfonamides and disulfonamides 

(5) Thiazides and hydrothiazides 

(6) Aldosterone antagonists and inhibitors of aldosterone secretion 

(7) OL /^-unsaturated ketone derivatives. 

The development of chlorothiazides and closely related compounds as diuretics 
and antihypertensive agents had its origin in the chemistry and renal physiology of 
sulfonamides and disulfonamides. The key compound in the initial work was sulfanila- 
mide. Domagk's (1935) observation that animals infected by the deadly hemolytic 
Streptococcus B survived when treated with Prontosil led Trefouel, et al. (1935), to 
study the structure-activity relationships of various analogues of this substance. Their 
observation that animals treated with Prontosil, 4'- sulfamyl-2, 4-diaminoazobenzene 



H-2 

excreted p-acetaminobenzenesulfonamide as a metabolite, proved that the active 
moiety of the sulfonamide dye was sulfanilamide. 

The key role of sulfanilamide in diuretic research began with a study of the side 
effects of sulfa drugs. Clinical studies revealed that some patients treated with sul- 
fanilamide developed acidosis, a condition in which the hydrogen ion concentration (pH) 
in the blood drops below the normal 7.35. Mann and Keilin (1940) discovered that this 
side effect resulted from the inhibition of carbonic anhydrase, the enzyme responsible 
for the conversion of carbon dioxide and water to hydrogen ion and bicarbonate ion. 
Pitts and Alexander (1945) were able to show that sulfanilamide acted as a carbonic 
anhydrase inhibitor, a condition which led to a decrease in the hydrogen-ion concentra- 
tion available for maintaining acid- base balance in the kidney. Consequently, inhibition 
of the enzyme carbonic anhydrase depresses the hydrogen- sodium exchange in the 
kidney and results in a more alkaline urine since a larger amount of sodium bicarbonate 
ions is excreted. 

Schwartz (1949) took advantage of the increased sodium excretion caused by sul- 
fanilamide to promote a diuretic action in patients suffering from congestive heart 
failure. He reasoned that the natriuretic effect of the compound should facilitate water 
excretion. Schwartz was able to show clinically that the diuretic action of sulfanilamide 
was due to its inhibition of carbonic anhydrase. He also observed that the chronic use 
of sulfanilamide led to excessive excretion of sodium and bicarbonate ions and con- 
comitant acidosis, rendering the carbonic anhydrase inhibitor ineffective for further 
diuretic action. Moreover, the amount of sulfanilamide necessary to produce a diuretic 
effect was too high and caused toxic side effects. 

In 1948 Krebs reported on his evaluation of sulfonamides as carbonic anhydrase 
inhibitors. He observed that an unsubstituted sulfamyl group is necessary for maximum 
activity. He also observed that high carbonic anhydrase inhibition was obtained when 
the unsubstituted sulfamyl group was attached directly to an aromatic ring. Especially 
significant was Schwartz's observation that acetylation of the amino group of sulfanila- 
mide resulted in an increase in activity over the parent compound. This structure/ 
activity discovery led to the preparation of acetazolamide by RobLin and Clapp (1950), 
of Lederle Laboratories, the first clinically effective nonmercurial orally active 
heterocyclic diuretic. The success with acetazolamide warranted study of the structure/ 
activity relationships of other heterocyclic sulfonamides, disulfamyl anilines, modified 
benzenesulfonamides, and heterocyclic mono- and disulfonamides. However, the greatest 
advancement in structure/activity relationship in this area was made when Sprague and 
Novello at the Merck, Sharp & Dohme Laboratories investigated ring closure studies 
starting with appropriately substituted 1, 3- benzenedisulfonamide. This led to their 
discovery (in 1957) of chlorothiazide 






02 

a major development in the search for nonmercurial diuretics. This was followed by 
the discovery of de Stevens, et al. ( 1958 and 1959), in the Ciba Laboratories, of hydro- 
chlorothiazide which was diuretically 10 to 15 times more potent than chlorothiazide. 
Both compounds received quick clinical acceptance. The development of chlorothiazide 



H-3 

and hydrochlorothiazide was followed by a new wave of research on structure/activity 
relationships that led to the discovery of thiazides whose diuretic potency varies over 
a range of 10, 000 fold. Demonstrated in this research on molecular modification was 
the significant increase in therapeutic ratio that results from the complete separation 
of carbonic anhydrase inhibition from the promotion of electrolyte excretion. 



HISTORICAL ACCOUNT 



Synthesis and Evaluation of Chlorothiazide 



Of all the sulfonamides, only unsubstituted sulfanilamide 




SO2NH2 



itself was a carbonic anhydrase inhibitor and diuretic. This suggests the need for a 
free primary amine group in the sulfonamide moiety. Contrasting requirements are 
found concerning the amino group para to the sulfonamide moiety. Here, free-NH-, is 
essential for the bacterial effect, but not for the diuretic action. Modification of 
structure to improve potency with respect to carbonic anhydrase inhibition led to hetero- 
cyclic sulfonamides like benzothiazole-2-sulfonamide 




SO2NH2 



which are hundreds of times more potent than sulfanilamide when tested against the 
enzyme in vitro. But this compound had no diuretic action when administered orally to 
dogs (Clapp, 1956). The 6-ethoxy derivative proved effective as a diuretic in animals 
and man (Miller, et al. , 1950; Roblin, et al. , 1950) but had adverse side effects. 
Further study showed that acetylation of the free amino substituent of a thiadiazole 
compound enhanced enzyme inhibition. Such a reaction produced acetazolamide (Diamox) 



n\i 



CH3C-N — l^v^ >— SOjNHj 



which was found to be 300 times more potent than sulfanilamide as a carbonic anhydrase 
inhibitor, and became the first oral sulfonamide derivative used as a diuretic. From 
1951-1958 it was chosen when a drug with a mild diuretic effect was desired. Diamox, 



H-4 

however, turned out to be of limited value, since tolerance and acidosis develop 
rapidly when it is administered for longer than 48 hours. Diamox is more useful as an 
adjunct to mercurial diuretics to restore or potentiate their effects, or to correct 
hypochloremic alkalosis resulting from their use. The carbonic anhydrase inhibiting 
effects of Diamox and its newer analogues ethoxyzolamide (Cardrase) and methozolamide 
(Meptazene) are used to control ocular tension by decreasing the rate of secretion of 
aqueous humor. The compounds also have found use in congestive heart failure asso- 
ciated with obstructive respiratory insufficiency and retention of carbon dioxide. 

The next effort in the search for better diuretics was to improve the assay proce- 
dure, paying less attention to carbonic anhydrase inhibition and more to actual in vivo 
diuretic activity. Data obtained in dogs with compounds related to acetazolamide are 
shown in Table H-1 in comparison with acetazolamide itself and with sulfanilamide 
(Beyer and Baer, 1961b). Pharmacological evaluation of diuretics is discussed in a 
later section of this appendix. 

The benzene disulfonamides turned out to be key compounds in the development 
of modern diuretic therapy. Much of the success along these lines can be attributed to 
the early findings of Krebs (1948), who noted that 4-carboxybenzenesulfonamide 

SO2NH2 



o 



COOH 

showed significantly high carbonic anhydrase inhibition. Very little was done with this 
published information until Beyer (1954) showed that this compound was capable of 
producing an appreciable natriuretic effect. This effect was accompanied by a small 
but significant increase in chloride excretion in the dog, an observation that was clini- 
cally corroborated by Lindsay and Brown (1954). However, Lindsay and Brown reported 
that 4-carboxybenzenesulfonamide was poorly absorbed upon oral administration and 
was relatively weak. On the basis of this slightly encouraging report, Sprague (chemis- 
try) and Beyer (renal pharmacology) merged efforts at Merck, Sharp & Dohme Labora- 
tories to explore this class of compounds in depth. They found in 1961 that 4-chloro- 
benzenesulfonamide had about the same enzyme inhibitory activity in vitro as sulfanila- 
mide and 4-carboxybenzenesulfonamide. Next, they investigated the 1, 3- benzenedisul- 
fonamides containing a chloro group on the benzene ring. Novello, et al. (1960), 
found that the compound 2, 4- bis(sulfamido)- chlorobenzene 




SO2NH2 



SO2NH2 

substantially increased the excretion of sodium, chloride, and bicarbonate ions when 
tested in experimental animals. Russo, et al. (1958), prepared dichlorphenamide 



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Ui <U B^ U 






-si 3 s " 

1) o j;-c E o 
c M 3 u-f; 5= I 






'-O'O 4J-S 

S a> — T3 «i a> " 
O J=— 'C Q-O « 

— c t: « aj — 

O.r a> 1> O f (a 

J; E h 3~.^ 

« J2 -^ — — -^ 

= »> - 2d.>. 
= 5^^ SOS 



jj a 



^ r; o 0^0 60^ 

JS u ^ U i_ S 
— to, ^w'flJ_g 

•^ «' -c M a = a 

^t: c £ > «T3 



,2 5; 



■2 S 



H-6 



CI 



H2NO2S ^\/^S02NH2 



and found that its renal action was quite similar to that of acetazolamide except for the 
excretion of less bicarbonate ions and more sodium and chloride ions. Clinical evalua- 
tion revealed that because of its greater chloruretic effect, dichlorphenamide causes 
less acidosis and shows greater tolerance than does acetazolamide. 

Sprague and co-workers prepared many compounds of the 1,3-benzene- 
disulfonamide class before they discovered 2, 4- bis{ sulfamido)- 5-chloroaniline benzene 



NH, 



H2NO2S 



o 



■S02NH2 



The rationale behind its synthesis may have been to prepare a compound which would 
incorporate the structural features of sulfanilamide and 2, 4-bis( sulfamido)-chloro- 
benzene with the chloro groups situated para and ortho, respectively, to the sulfamyl 
groups. This rationale is reminiscent of some of the generalizations made by Krebs 
(1948). Sprague (1957) reported that the compound produced a pattern of sodium 
chloride excretion somewhat different from that of the carbonic anhydrase inhibitors 
and more like that of the organic mercurials. Lund and Storling (1959) confirmed this 
saluretic response in many derivatives. 

At this stage, the search for diuretics took two divergent courses: 

(1) The preparation and evaluation of additional 1, 3-benzenedisul- 
fonamides, modified benzene sulfonamides, disulfamyl anilines, 
and heterocyclic mono- and disulfonamides 

(2) The conversion of 2, 4- bis(sulfamido) -chlorobenzene into the 
cyclic derivative 6-chloro-7-sulfamyl-2H- 1, 2, 4- benzothiadia- 
zine-1, l-dioxide (chlorothiazide) i 

The ring substituted 1 , 3 -benzenedisulfonamides studied initially by Sprague and co- 
workers were shown by Beyer and his group to possess diuretic properties of consider- 
able interest. In addition, the N-acyl (CH3CO to CgH^^CO) derivatives of 2, 4- bis 
(sulfamido)-chlorobenzene were more active than the parent compound, the diuretic 
activity increasing from CH3CO to n-C^H^jCO. However, these compounds were effec- 
tive clinically only at high doses, and avoidance of acidosis was questionable. 

A detailed study of the classes of compounds identified under (1) above still left 
the preparation of an effective diuretic agent unsolved. Then, Novello and Sprague (1957) 
struck on a most important development. Following a procedure first described by 
Ekbom (1902) on the condensation of 2- sulfamidoaniline with formic acid, they treated 
5-chloro-2, 4- bis (sulfamido) aniline with formic acid to obtain the unstable formylamino 
intermediate which yielded chlorothiazide; 



H-7 



H2NO2S 



.NH- 



o 



HCOOH 



•SO2NH2 



o 



H2N02S 



•S02NH2 



CI. 



HgNOgS 




o 



I 
NH 



Chlorothiazide's outstanding saluretic effect in animals prompted immediate 
clinical evaluation. The compound showed an unusually high degree of diuretic activity 
with low toxicity when tested in humans. The daily oral dose was found to be 500 to 
2000 mg. Chlorothiazide was less than 1/20 as potent an inhibitor of carbonic anhydrase 
as acetazolamide (although still 10 times more potent than sulfanilamide). It was 5 to 
10 times more potent than acetazolamide in promoting the loss of sodium and chloride. 
Within 2 years it had become accepted as an effective oral diuretic. Merck, Sharp & 
Dohme received FDA approval for diuretic use of chlorothiazide (Diuril NDA- 1 1- 145) 
on October 4, 1957. 

Chlorothiazide, however, was less potent than acetazolamide in its saluretic effect. 
It was also clear by now that omission of the chloride group in chlorothiazide abolished 
chloruresis. Poorly understood also at this stage were the possible interrelationships 
between therapeutic ratio, carbonic anhydrase inhibition, and promotion of electrolyte 
excretion. This lack of information resulted in an avalanche of work in which chemi- 
cally and biologically heterocycles based on chlorothiazide were synthesized and 
evaluated for diuretic properties. The approaches to syntheses can be illustrated by 
examining some of the possible structural modifications of chlorothiazide 



HgNOjS 




(1) Reduction by addition of hydrogen at the 3,4 linkage 

(2) Replacement of the chloride group in the 6 position by another 
electronegative group 

(3) Substitution of different organic radicals for the hydrogen at 
the 3 position 

(4) Substitutiftg an alkyl group or some other radical at the 
2 position of the molecule. 



H-8 
Derivative s of Chlorothiazide 

ChLorothiazide marked the great breakthrough in the search for noamercuriaL 
diuretics. However, subsequent research quickly indicated that experimental conditions 
to optimize structure/activity relationships of potential diuretic materials still were 
not clearly understood. For example, it became of interest to determine the effect of 
pH on the nature of products derived from the condensation of sulfonamides with alde- 
hydes. The formation of polymeric materials had been reported. However, Freeman 
and Wagner (1951) observed that substituted o-aminobenzenesuLfonamides react with 
formaldehyde in alkaline solution to yield R-N(2)-3, 4-dihydro 1, 2, 4-benzothiadiazine- 1, 
1 dioxide (R = phenyl or substituted phenyl). 

De Stevens, et al. (19 58), carried out the same reaction on unsubstituted o-amino- 
benzenesulfonamide and with various aldehydes in nonpolar solvents containing catalytic 
amounts of hydrogen chloride, and analogous compounds were obtained. 

Next, attention was directed to the condensation of 2, 4- bis(sulfamido)- 5-chloro- 
aniline with various aldehydes. With formaldehyde under acidic conditions, cyclization 
yielded 6-chloro-3, 4-dihydro-7- sulfamyl-2 H-1, 2, 4-benzothiadiazine 1, 1-dioxide 
(hydrochlorothiazide). This structure was confirmed by sodium borohydride reduction 
of chlorothiazide to hydrochlorothiazide, and by reconverting the hydrochlorothiazide 
to chlorothiazide via permanganate oxidation. Hydrochlorothiazide was found to be 
from 10 to 15 times more potent than chlorothiazide as a diuretic when tested in dogs 
and humans. The observation of the increased activity of the dihydro compound was 
confirmed in many hundreds of compounds prepared in this series for diuretic evalua- 
tion. The discovery of hydrochlorothiazide was a major triumph in this field after the 
discovery of chlorothiazide by Novello and Sprague. 

The success with hydrochlorothiazide triggered the chemical synthesis, pharma- 
cological evaluation, and clinical evaluation of a large number of chlorothiazide deriva- 
tives with various structural and activity characteristics. 

The relationship between structure and activity is complex and depends not only 
on the chemical configuration of the compound but also on many physiological factors 
such as route of administration, rate of drug excretion, test assay system, and degree 
of edema or electrolyte imbalance. Several generalizations can be made: 

(1) Any stable substitution on the 7- sulfonamide group destroys carbonic 
anhydrase inhibitory activity. 

(2) Substitutions at the same position decrease chloruretic activity but 
do not abolish it (Maren and Wiley, 1964), although the exact activity 
of N-7 acetyl derivatives is complicated by in vivo deacetylation 
(Duggan, 1967). 

(3) There is no correlation between in vitro carbonic anhydrase and 
in vivo chloruretic response. 

(4) Various halogen substitutions at the 6 position enhance chloruretic 
activity. 

(5) Saturation of the heterocyclic ring between the 3 and 4 positions also 
increases potency, for example, of hydrochlorothiazide. 



H-9 

(6) A variety of substituents at the 3 position increase diuretic potency. 

(7) The addition of various functionaL groups at the 1, 4, and 5 positions 
increases diuretic activity. 



Chlorthalidone, 




SOzNHg 




H 



and quinethazone, 

H2NO2S 



are sulfonamide diuretics v/hich differ chemically from the thiazides by the nature of 
the heterocyclic ring. However their pharmacological action is similar to that of 
thiazides. Chlorthalidone was prepared by Graf et al. (I960) at the Geigy Laboratories 
and quinethazone by Cohen et al. (1959) at the Lederle Laboratories. 

All thiazides thus far discussed have parallel dose- response curves and compar- 
able maximal chloruretic effects. This suggests that they have a similar mechanism 
of action. The various thiazide analogues differ primarily in the dose required to 
produce a given effect and do not necessarily differ with respect to their maximal 
experimental effect or optimum therapeutic response. The maximum diuresis produced 
by large doses of the weakest member of the group cannot be exceeded at any dose by 
the most potent member. Comparative potencies may vary some, depending on the 
assay system. In general, data obtained in the dog and in clinical trials in man are in 
reasonable agreement. 

Pharmacological Evaluation of Diuretics 



Lipschitz, et al. (1943), used the rat as the animal model for the evaluation of 
diuretics. They fed small volumes of 0.9 percent sodium chloride solution to rats and 
then tested urea, saline diuretics, xanthine derivatives, and mercurials. They cal- 
culated the relative efficiency of the drugs by plotting their dose-effect curves against 
that of urea. The sequence of potencies turned out to be the same as that established 
in man. De Stevens, et al. , found that the rat test served as a useful rapid screening 
method for the selection of candidate diuretics to be tested in dogs. 

They selected unanesthetized dogs as the animal model for the detailed evaluation 
of diuretic agents because the functional resemblance of the dog and human kidneys 
justifies a broad exchange of data between these two species. Normal renal functions 
and response to diuretic drugs are almost identical for dog and man when corrected 
for differences in kidney weight. Glomerular filtration rate, renal blood flow, ion 



H-10 

excretion, and ability to concentrate and dilute urine of the two species show excellent 
agreement. The major differences between the kidney of the dog and man lie in their 
response to disease and in their method of handling a salt load. The latter is pertinent 
to the chlorothiazide study. Oral or intravenous intake of several grams of salt by a 
human is followed by a slow (about 3 days) renal excretion of the load. This process 
seems to be mediated by the adrenal cortex through the renal effect of slight inhibition 
of tubular reabsorption. By contrast, the dog responds to a salt load with a rapid in- 
crease in glomerular filtration rate and elimination of the salt load is complete within 
several hours. These contrasting mechanisms for salt excretion have no .bearing on 
the problem at hand. Diuretics act by inhibiting the reabsorption of sodium in the renal 
tubules resulting in the increased urinary excretion of both sodium and water. Re- 
sponses to this drug by man and by dog, his substitute subject for experimentation, are 
parallel. 

In a typical experiment at the Ciba Laboratories, the prescreening observation 
was made on male rats, fasted 18 hours, and given 5 ml per 100 g body weight of 0.2 
to 0.9 percent sodium chloride solution by stomach tube. The rats were placed in 
metabolism cages and urine volumes were measured at 30-minute intervals over a 
3-hour period. The unanesthetized, fasted dogs received a subcutaneous injection of 
100 ml of 0.9 percent saline. Next, they received the candidate diuretic orally or by 
"injection. Urine was then collected by catheterization at intervals of 2, 4, and 6 hours 
after the administration of the drug. Urinary sodium and potassium concentrations 
were measured by means of a Bair flame photometer and expressed in milliequivalents 
of sodium and potassium excreted in each of the three successive 2-hour intervals. 
Urinary chloride was analyzed polarographically. The results of such an experiment 
were expressed as the average volume of urine in milliliters and average milliequiva- 
lents of electrolyte excreted per dog in the 6-hour period or for each 2-hour interval. 
The saline controls were run prior to the drug experiments and also at intervals during 
the course of the drug studies, 

Beyer (1961a) evaluated the efficacy of some compounds in dogs made edematous 
by giving them 9 a-fluorohydrocortisone, sodium chloride, potassium chloride, and 
water. At this stage, they were treated with the diuretic agent to effect water diuresis 
to the level of healthy controls. 

Fuchs (I960) reported the clinical pharmacology of hydrochlorothiazide. In a 
2-year study, it was administered successfully in edemas caused by cardiac insuffi- 
ciency, cirrhoses, toxicoses of pregnancy, and nephroses. Effective dosage was estab- 
lished at 25 to 200 mg daily. Diuresis occurs within a few hours and lasts at least 
12 hours. Hydrochlorothiazide is well tolerated and such secondary effects as hypo- 
natremia, azotemia, and hyperuricosemia are rarely observed. Clinical evaluations 
of hydrochlorothiazide were also conducted by Utley, et al. (1961), and Waugh, et al. 
(1961), who both confirmed Fuch's findings. 

Rosenblum (1961) found that hydrochlorothiazide at an oral dosage of 50 to 100 mg 
twice a day is an effective diuretic in the treatment of patients with edema of cardiac 
and hepatic origin. Of the 26 patients treated, there was no significant alteration of 
the serum electrolyte pattern in 24. Two patients with Laennec's cirrhosis developed 
prehepatic coma while on hydrochlorothiazide and supplemental potassium therapy. 

Caliva, et al. (I960), reported their clinical evaluation of new saluretic agent, 
hydroflumethiazide. The diuretic was administered in doses of 50 mg twice daily to 
29 hospitalized patients and 45 clinic patients. Of these, 48 showed overt signs of 



H-11 

fLuid retention, and 28 did not. Hydroflumethiazide effects an increased excretion of 
water, sodium, chloride, and potassium. Serum electrolytes are essentially unchanged, 
although there is a trend toward lower chloride and potassium values. In 26 hyperten- 
sive outpatients who were followed for approximately 12 weeks, the average fall in the 
mean blood pressure was 12 mm Hg. Sixteen of these patients previously had been 
receiving chlorothiazide, or its analogue, and in 6 of these, there was a further drop 
in blood pressure when they received hydroflumethiazide. 

Fuchs, et al. (1961), found trichlormethiazide 



CI 



HjN OgS 




o 



XHCIj 



.NH 



02 



to have actions similar to chlorothiazide except that it was effective at much lower 
dosages (2 to 4 mg) daily. Doses greater than 8 mg have no further effects on electro- 
lyte excretion. The results of Schafer's clinical study (1961) of trichlormethiazide 
suggested that electrolyte excretion was more favorable than with other benzothiadiazine 
derivatives. 

Clinical studies of Ford (1961) indicate that polythiazide 



CI 



HgNOjS 




o 



S' 
02 



^CHj-SCHgCFj 
-N-CHs 



has a predominant effect on the excretion of sodium and a lesser effect on potassium 
excretion. It is 2. 1 times as potent following oral administration as meralluride, 
following parenteral administration. When used in chronic therapy of patients with 
edema of varied etiology, there are no significant changes in serum biochemistry. 
Polythiazide is also effective in the management of hypertensive states and has a dura- 
tion of action of at least 36 hours. 



Ford (1961) found hydrobenzthiazide 



CI 



HgN OgS 




o 



,CH2 



-SCH.^gj) 



02 



NH 



an effective saluretic agent with moderate potassium loss. It is repetitively effective 
in edematous states of varied etiology and as an adjunct to antihypertensive 
regimens. 



H-12 







H 
1 


HaNOzS-^ 


@ 


O2 



MethycLothiazide 



(Ford, I960) is an effective diuretic with diuresis occurring within 2 hours, peaking 
within 6 hours, and lasting more than 24 hours. It is aLso effective in a wide variety of 
edematous and hypertensive states and upon continuous administration causes no signifi- 
cant changes in serum biochemistry. MethyLclothiazide significantly increases natri- 
uresis and Lessens kaliuresis. 



Adverse Reactions of Thiazides 



The single most important complication of therapy with thiazides is a secondary 
kaliuresis and potassium depletion. This complication may be avoided by dietary sup- 
plementation or the simultaneous use of an aldosterone antagonist. Mild, untoward 
effects such as gastrointestinal disorders, dizziness, weakness, and fatigue generally 
inay be controlled by reducing the dose. 

Renal tubular reabsorption of uric acid may lead to elevated uric acid levels in 
the blood. Asyinptoniatic hyperuricemia has been observed, but acute gouty arthritis 
has occurred only infrequently, except in individuals with a presumed hereditary dis- 
position to gout or Ln patients with chronic renal failure. 

The development of hyperglycemia and aggravation of preexisting diabetes noted 
occasionally with the thiazides are usually reversible upon discontinuation of the drug. 
Increased amounts of hypoglycemic agents inay be needed to control the elevated blood- 
sugar concentrations. 

Hypotensive effects under anesthesia have been observed in some patients receiv- 
ing thiazides. To avoid a negative potassium balance, the thiazides are often pre- 
scribed in combination with potassiuni chloride. The use of enteric-coated combinations 
of potassium chloride and thiazide has been associated with a suspiciously high incidence 
of ulceration of the distal jejunum or ileum. 



Therapeutic Uses of Thiazides 



These agents are used to initiate diuresis and maintain the edema-free state in 
cardiac decompensation. The diuretic effect is dependent upon sufficient glomerular 
filtration of sodium and is reduced or nullified in the presence of severe renal diseases 
when the glonierular filtration rate is greatly reduced. The oral thiazides often can be 
used in place of the organic mercurials which must be administered parenterally. 

The thiazides and related compounds also have been used in patients with edema 
associated with nephrosis and certain types of nephritis, liver disease, pregnancy, and 
the premenstrual syndrome, as well as edema induced by the administration of corti- 
cotropin, adrenal corticosteroids , and some estrogens. However, they have been less 



H-13 

effective in therapy for these conditions than in therapy for congestive heart failure. 
The thiazides also can be used alone or in combination with other drugs for the manage- 
ment of hypertension. 

Although the effect is only palliative, the oral diuretics reduce the polyuria of 
nephrogenic and nonnephrogenic diabetes insipidus; in infants and young children, they 
may be of special value in the prevention of dehydration, hyperthermia, and other 
complications of these diseases. 



REFERENCES 



Beyer, K. H. , Peters, H. F., Patch, E. A., and Russo, H. F. , J. Pharmacol., _82, 
239 (1944). 

Beyer, K. H., Russo, H. F. , Patch, E. A,, Peters, L. , and Sprague, K. L. , J. Lab. 
Clin. Med., 3J., 65 (1946). 

Beyer, K. H. , McKinley, S. E., Tillson, E. K,, and Green, C. W., J. Pharmacol. 
Exptl. Therap., ^, 263 (1947). 

Beyer, K. H., Arch. Intern. Pharmacodyn. , _98, 17 (1954). 

Beyer, K. H. , and Baer, J. E., Progressive Drug Research, E. Jucker (Ed.), II, 10, 
Berkhauser Verlag, Basel, Switzerland (1961a). 

Beyer, K. H. , and Baer, J. E., Pharmacol. Rev. , _n, 517 (1961b). 

Beyer, K. H. , Sprague, J. M. , Baer, J. E., andNoveUo, F. C., Medical World 
News, 57 (November 17, 1975). 

Caliva, F. S., Taylor, F., Chodos, R., Chaffee, W., and Lyons, R. , New York J. 
Med., 4016 (I960). 

Clapp, J. W., J. Biol. Chem., 223, 207 (1956). 

Cohen, E., Klarberg, B. , and Vaughn, J. R. , J. Am. Chem. Soc, ^, 5508(1959). 

Davenport, H. W., andWilhelmi, A. E., Proc. Soc. Exper. Biol. & Med., £8, 53 
(1941). 

de Stevens, G., Werner, L. H., Halamandario, A., and Ricca, S., Experientia, 14, 
463 (1958). 

de Stevens, G., and Werner, L. H. , Swiss Patent 337, 205 (May 1 5, 1959). 

de Stevens, G. , Diuretics Chemistry and Pharmacology, Academic Press, New York 
and London (1963). 

Domagk, G., Deut. Med. Wochschr., 6J., 250 (1935). 

Duggan, D. E., J. Pharm. Exp. Therap., J^, 193 (1967). 

Ekbom, A., Bih. Svenska, Vet. -Akad. Handl. [ II] , 2_7 (1), 22 (1902); Beilstein, 4th ed. , 
27, 570; 14, 682 (1918). 

Ford, R. v., Southern Med. J., ^, 40 (1959). 

Ford, R. v.. Current Therap. Res., 2, 422(1960). 

Ford, R. v., Current Therap. Res., 3, 320(1961). 



H-14 

Freeman, J. H. , and Wagner, E. C, J. Org. Chem. , J_6, 815(1951). 

Fuchs, M., Chemotherapia, j., 231 (1960). 

Fuchs, M., Newman, B. E., and Seller, R. H. , Current Therap. Res., 3, 117(1961). 

Goodman, L. S. , and Oilman, A., The Pharmacological Basis of Therapeutics , 4th 

Ed., 855, The MacMillan Company (1970). 

Oraf, W., Oirod, E., Schmid, E., and Stoll, W. O. , Helv. Chem. Acta, 42, 1085 

(1959). 

Krebs, H. A., Biochem. J., 43, 525 (1948). 

Lindsay, A. E., and Brown, H., J. Lab. Clin. Med., 43, 839 (1954). 

Lipschitz, W. L. , Hadidian, Z., and Kerpscar, A. J., J. Pharm. Exp. Therap., T^_, 

97 (1943). 

Lund, A., and Storling, Acta Pharmacol. Toxicol., j_5, 300 (1959). 

Mann, T., and Keilin, D., Nature, 146 , 164(1940). 

Maren, T. H., and Wiley, C. E., J. Pharm. Exp. Therap., }A^, 230(1964). 

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(1950).' 

Moore, M. L. , and Miller, C. S. , J. Am. Chem. Soc, _63, 2781 (1941). 

Novello, F. C, and Sprague, J. M., J. Am. Chem. Soc, ^^_, 2028(1957). 

Novello, F. C. (to Merck and Co. , Inc.), U.S. Patent 2, 809, 194 (October 8, 1957). 

Novello, F. C, Bell, S. C, Abrams, E.L.A., Ziegler, C, and Sprague, J. M., 

J. Org. Chem., 25, 965 (I960). 

Pitts, R. F., and Alexander, R. S., Am. J. Physiol., j_44, 239 (1945). 

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Rosenblum, M. A. , New York State J. Med., 3417 (1961). 

Roughton, F.J.W., Physiol. Rev., \5_, 241 (1935). 

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r?, 407 (1958). 

Schaefer, L. C, Clin. Med., 1343 (1961). 

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1714 (1940). 

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(1946). 

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H-15 

Sprague, J. M. , and Miller, C. S. (to Sharp & Dohme, Inc.), U. S. Patent 2, 608, 506 
(August 26, 1952). 

Sprague, J. M., N.Y. Acad. Sci. , 7J., 328(1957). 

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120 , 756 (1935). 

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Waugh, B. S., Kain, T. M. , and Ruckstuhl, L., J. Med. Soc, N. J., 101(1961). 



Carbonic Anhydrase Enzyme Studies 



Aromat 



1935 Roughton T 



1935 Domag 



1935 Trr 



191! 



i94r 



Carbonic Anhydrase Enzyme Studies 



Aromatic and Heterocyclic Sulfonamide Studies 



Chlorothiazides 



1936 Roughlon Oisco\*ied Ihe etiivme car- 
NR/0/W/P/M 

1935 Oomagk Obierved thai animals infected 
with Sireptococeui B survived 
when Heated with Pfontojil B 

I93S Treloue! Found that animali tteaied 
with Piomoiil B eicreted 
P-aceia-artiidobenicne- 
sullonamide as a metabolite 
NR/S/W/P/M 

1940 Mann Some patienij Heated with lul- 
fan'lamide developed acidotit- 
caused bv inhibil'oo of 
carbonic anhvdraie enlyfo 

NR/S/W/P/M 

. 1941 Davenport Found high concenuotions 
oi carbonic anhydraie in 
the cotlBX ol cat, dog, and 



cidflted cause o( aci 
IMR/S/U/P/M 



1918 Ekbom Fleaciion ol lormic acid ^vnh 
2aminobcnjenesul(onamidB 
yielded 1,2,a-benioihLadia- 
line-l.l-dioKido 

NR/S/W/PM 

1940 Sprague, Sobslituted luHamla- 

mides 
NR/S/U/P/C 

1941 Spraflue. Sullonaniidoj of 



1941 Moore, Sultonamidoihiaiolonej 

NR/S/U/PC 

1943 Sharp & Initiated baiic renal retearch 
Dohme program headed bv Beyar 

and Sprague 

MR/0/U/P/C 

1944 Boyer, Renal elimination of sullamera- 

line, sullamothazinc. luKadia- 
line, and sullaihiaiole by the 



194G Bayei, Ft 



■tylated suHonemidei 
MR/S/U/P/C 



1946 Sprague. CarbOHy dorivaliv^i ot luKon- 
amidothiaiolei 

MR/S/U/P/C 



1947 Beyei. Renal clearance siudiej on 4'- 
carbo'typhGoylmethanesuKon- 



MR/S/U/P/C 



inhibition of tatbonic anhydraie 
NR/S/W/P/M 

1948 Krebs Found Ihai monoacety'ation o( 
the ammo group ot sulfanilamide 
increased inhibition aaivity over 



1 Schtwriz Used sulfanilamide to promote 
diuresis in patients suffering 
Irom congestive heati lailure. 
Showed that diuret.c action 



n of c 



jnhydra 



1949 Schwari? Repoftad adi/flrsa effect* on 

chronic use of tulranilamida ~ 
toxicity, eicaaivB cucteiion 

ion I with concomitar)! 



1949 Sprague. 2-SuHanitamido-4. S-dicarboiy 

thiaiole 

MR/S/U/P/C 

1950 Sprague, NIsubstnuied sulfonyH ammo- 

benzoic acids 

MR/S/U/P/C 

1950 Roblin Prepaied acflla/olamide - the 
(irst clinically effective non- 
mercurial orally active heieio- 
cvellc diuretic. Prolonged usage 
leads to acidosis and pro- 
nounced potassium depletion 
MR/S/U/P/C 

1952 Sprague. (Atyl lulfamyll benjoic acids 
MR/S/U/P/C 



COOES 

No nmission-Orie filed Research 

Mission-Oriented Research 

Developmeni 

Significant Event 

Decisive Eveni 

U.S. Event 

Foreign Event 

Government Funding 

Private Funding 

Medical School - Hospital - 

University 

Research Institution 

Government Laboratory 

Company 



19S4 Beyer Demonstrated thai 4^arboxv- 
beniene-tulfonamide pottetsed 
sinnificani natriuretic pfopcnies 



1954 Lindsay 



Corroborated Beyer's obier. 
vation (19641 but observed 
compound to be poorly 
absorbed upon oral 

MR/S/U/P/C 



1956/ Spraguo, 2,4-Bis I(u1fflmido)-Sehloroani- 
1957 line found io be eKecllvc sal- 

uretic agant in dogi 
MR/D/U/P/C 

1956/ Lund, Confirmed clinical oMecliveness 
1957 of 2.4-6is (sulfamidol-5<:hloro- 

MR/S/U/P/C 

1957 Spiasuo, Acylation of 2.A-b\t liulfsmidol 
5«htoraniline gave compounds 
in which diuretic activity in- 
creased m going from CHjCO- 
toCcH,,CO 



MR/S/U/P/C 



1957 NovBllo Heaciionol 2.4.bijlwlf- 

amidol-S^hloroanillno wit 



MR/0/U/P/C 

19S7 Ford, PrBliminotv cllhiM' 



h 



HISTORIOGRAPH - CHLOROTHIAZIDE 



Ide piesenied at Southern 
Sociaiv for Clinioil Rtseorci. 
1-26-57 

MR/D/U/P/C 

1957 Novello Use ol twrnolhiadndn* dwoidn 
MR/D/U/P/C 

1957 M«rek, Siarp Use ot ehroroihiatrie ai 

a Dohma diuratic approved by FDA 
10/57 IDiuril I1-U5I 
MR/D/U/P/C 

1969 deSiovens. HydrobeniothtKJiajinB dio»- 
>d«i iMih potent dmralie 
etieci 

MR/D/U/P/C 

1959 deSlewns, Hydrochlorolhiaii* 
Mfl/D/W/P/C 

1975 8«YBr. Sprague. Shared a S10.000 

Bmt a Nov«1k> Altwi L«ik«' Spacul 
Award Mvaring 
cniorothiUMte 



I-l 



APPENDIX I. COMBINATION CHEMOTHERAPY OF 
ACUTE CHILDHOOD LEUKEMIA 



INTRODUCTION AND SUMMARY 



The term, "acute Leukemia" refers to a type of leukemia which, without treatment, 
runs a rapidly progressive and ultimately fatal course. Since the advent of chemo- 
therapy, however, remission from the disease can often be accomplished. This 
results in a syndrome characterized by periods of remission of varying lengths often 
with intervening periods of relapse. Therefore, the term "acute leukemia" now is used 
to refer to those syndromes in which there is a predominance of immature leukemic 
cells which are morphologically similar to normal blast cells rather than referring to 
the duration of the clinical syndrome (Freireich and Frei, 1964). 

The recognition that human cancer could be treated by chemotherapeutic agents 
was attributable to wartime research with nitrogen mustards as reported by Rhoads in 
1946, although the first significant demonstration of beneficial effects was reported by 
Farber in 1948. Laboratory studies were immediately initiated in an attempt to find 
more useful drugs of the same classes as those described by Rhoads and Farber (i.e., 
alkylating agents and folic acid antagonists). Clinical studies were employed in an 
attempt to develop a treatment regimen which would successfully control human cancer. 
Early successes were meager and the use of these agents was restricted to a relatively 
few clinicians. However, as new agents and new classes of agents were described for 
cancer therapy, successes in remission induction were more encouraging. Early 
cancer chemotherapy was primarily used against leukemias and it was soon recognized 
that acute lymphoblastic leukemia of children responded more favorably than did other 
malignancies. 

It was well known that combination chemotherapy was beneficial in the treatment 
of bacterial diseases. It was also well established that bacteria could and often did 
develop resistance against chemotherapy agents, and that the use of more than one 
drug could successfully circumvent such resistance. This was clearly shown to be the 
case for tuberculosis. This stimulated both experimentalists and clinicians to try 
combinations of chemotherapy agents to produce more effective remissions in animal 
and human cancer. By the early nineteen sixties, the increased effectiveness of com- 
bination over single-agent therapy had been demonstrated. 

By this time, there was an extensive amount of literature concerning pharmia- 
cologic studies, kinetics of leukemic cells, origins and mechanisms of resistance, use 
of single and combination chemotherapy in experimental tumor models, and biochemical 
aspects of new and existing classes of agents. By using the knowledge gained from 
these and prior clinical studies, new combinations of agents and new treatmient regi- 
mens were more rationally conceived and implemented. This resulted in progressively 
more successful therapy of childhood acute leukemia, so that by 1970 there was a 
number of reports of long-term remissions (5 years or more). By this time, combina- 
tion chemotherapy was well accepted as the most effective means available to prolong 
life in children afflicted with this disease. 



1-2 
HISTORICAL ACCOUNT 



Accession of Agents for Early 
Combination Chemotherapy Studies 



The era of modern cancer chemotherapy probably had its origin in the work of 
Huggins and Hodge in 1941 in which they studied the effects of castration, androgen 
injection, and estrogen injection on serum phosphatase levels in metastatic prostatic 
carcinoma (Zubrod, 1966). Results of the wartime evaluation of nitrogen mustards in 
the treatment of neoplastic disease reported by Rhoads in 1946 provided the initial 
impetus for much more extensive work on this and other alkylating agents in succeeding 
years. 

From that time, a steady procession of new cancer chemotherapy agents has been 
introduced into experimental studies and subsequently into clinical trials. Much of the 
earlier work on single-agent and combination chemotherapy of childhood acute lympho- 
cytic leukemia (ALL) was carried out using one or more of five drugs which were capa- 
ble of inducing complete remissions in this disease in children (Frei, 1965; Zubrod, 
1965). For this reason, the historical events relating to the introduction of these five 
agents (methotrexate, adrenal cortical steroids and ACTH, 6-mercaptopurine, vin- 
cristine, and cyclophosphamide) are now considered. 

During 1947 and 1948, Farber, et al. demonstrated that it was possible to achieve 
remissions in children with acute Lymphoblastic leukemia by the use of aminopterin, a 
folic acid antagonist. This was the first significant evidence that chemotherapy could 
be used advantageously in this disease. A new folic acid antagonist, amethopterin 
(methotrexate), was then reported in 1948 by Smith. Beneficial results were demon- 
strated against leukemia in mice by Burchenal, 1949; Law, 1952; and Stock, 1950. 
This drug was subsequently shown to be effective against human leukemia and to induce 
less toxicity than aminopterin (Burchanel, 1951; and Farber, 1951). 

Heilman and Kendall in 1944 showed that 1 1-dehyro- 17-deoxycorticosterone 
(compound E) would, when administered by oral or parenteral inoculations to mice, 
inhibit the growth or cause regression of transplantable tumors derived from an undif- 
ferentiated spontaneous neoplasm from the thoracic cavity of a mouse. Clinical efficacy 
of agents which induce similar activity was reported by Pearson, et al. , in 1950, when 
they demonstrated beneficial effects of ACTH in the treatment of chronic lymphocytic 
and acute lymphoblastic leukemias. Farber, et al. , in the same year, demonstrated 
efficacy of both ACTH and cortisone in the treatment of acute lymphoblastic leukemia. 

The efficacy of 6-mercaptopurine (6-MP) against acute Lymphoblastic leukemia 
and several other kinds of Leukemia was documented by Burchenal, et al. , in 1953. 
These studies were conducted in patients with no previous treatment and in some in 
which there was resistance to methotrexate or steroids. Cyclophosphamide was shown 
to be effective in treating ALL as reported by Fernbach in 1962. Cyclophosphamide 
was more useful in inducing remissions than were other alkylating agents which had 
been previously used. 

Clinical trials with vincristine in 1962 showed this drug to be effective in produc- 
ing remissions of ALL in children who had relapsed following earlier treatment with 



1-3 



other antileukemic agents (Selawry and Delta). In the following year, efficacy of this 
drug in the treatment of children with previously untreated cases of ALL was demon- 
strated by Evans, et al. It is of interest that the pharmaceutical firm which produced 
vincristine had decided to stop production of the agent until clinicians from NCI con- 
vinced them that it was a potentially valuable drug for cancer chemotherapy. This 
evidence was based on work at NCI which preceded the clinical trials described above 
(Zubrod, personal communication). 



Experimental Models in Studying Concepts of Chemotherapy, 
Especially as Related to Combination Chemotherapy 



Experimental animal-tumor models have had profound impact on the developmient 
of cancer chemotherapy and more specifically combination chemotherapy of acute 
childhood leukemia. Numerous such models have been developed. However, the L1210 
murine leukemia model first described by Law in 1949 has probably been the most 
significant for combination chemotherapy of ALL since most of the testing of drug com- 
binations for chemotherapeutic efficacy has been carried out in this model (Goldin, 
et al., 1971). 

Durth and Kahn, 1937, by the use of micromanipulation techniques, were able to 
perform inoculations of single cells from an experimental murine leukemia into mice 
of the same inbred strain. Of 97 mice which were injected, five developed leukemia 
and died within 15 to 50 days. This demonstrated conclusively that a single leukemic 
cell was capable of inducing the disease. This fact was of great significance in the 
later development of a philosophy of therapy for which the objective was total eradica- 
tion of all leukemic cells. This has special significance in considering the impact of 
drug resistance on the problem of developing chemotherapeutic regimens capable of 
producing a cure in ALL and other malignancies. 

Development of resistance to folic acid antagonists in transplantable murine 
leukemia models was demonstrated in 1950 by Burchenal, et al. , and Law and Boyle. 
Further experiments by Law in 1952 demonstrated that mutation and selection con- 
stitute the mechanism by which L1210 leukemia developed resistance to amethopterin. 

Goldin, 1956, using the L1210 model and Skipper, et al. , 1957, using the L4946 
leukemic model observed a correlation between the number of leukemic cells inoculated 
and the curability rate achieved with amethopterin. These results were interpreted to 
suggest the development of mutant strains of leukemic cells which were resistant to 
amethopterin. On the basis of these studies. Skipper suggested that since drug-resis- 
tant populations of leukemia cells appeared to be a limiting factor in the curability of 
mouse leukemia, then the approach to therapy should include combinations of agents 
that act at different biochemical sites and thus prevent cross resistance. 

Studies reported by Goldin et al. (1956) illustrate that the optimal therapy schedule 
and optimal dose may be influenced by the stage of disease, at least in the L1210 leuke- 
mia miodel. This group using amethopterin demonstrated that advanced L1210 leukemia 
was susceptible to therapy if appropriate dosages and schedules were employed. 

It was demonstrated by Brockman et al. , in 1958, that C -labeled azaguanine 
was not taken up by azaguanine- resistant L1210 cells developed by Law, while aza- 
guanine- sensitive cells became markedly labeled. This resistance may have been due 



1-4 

to failure of development of an enzyme in the resistant cells which is capable of con- 
verting 8-azaguanine to 8-azaguanilic acid. Brockman et al. (1959) further demon- 
strated the resistance of Streptococcus faecalis to 8-azaguanine. They concluded that to be- 
come inhibitory, 8-azaguanine must first be metabolized to 8-azaguanilic acid which 
then intereferes with nucleotide metabolism by being incorporated into nucleic acids or 
by forming a fraudulent cofactor. These studies were significant in developing early 
knowledge on mechanisms of drug resistance in chemotherapy. 

Skipper et al. , in 1964, studied the kinetics of leukemic cells in the L.1210 mouse 
leukemia system. It was shown that the size of inoculum of leukemic cells determine 
time to death, and that the percentage of leukemic cells killed is constant at a given 
drug dosage irrespective of the size of the inoculum. The latter observation was initially 
demonstrated by Golden, et al. , in 1956. It was further demonstrated by Skipper that 
the average generation time of leukemic cells in the L1210 model is approximately 
12 hours, and that following a 2-day lag phase after inoculation, a logarithmic proli- 
feration occurs until the lethal number of leukemic cells has been reached. These 
data were then used to estimate the percentage reduction of the leukemic-cell popula- 
tion in experimental systems and later in clinical trials with combination chemotherapy. 

Schabel in 1968 demonstrated that l-(2-chloroethyl)-3-cyclohexyl- 1 -nitrosourea 
(CCNU) and ARA-C, when administered in an appropriate schedule, resulted in long- 
term survival in 40 percent of mice in which 10^ Lil210 cells were given I.V. Neither 
agent alone was capable of producing survivors in the experimental regimens used. 
The success of the combination was probably due to the eradication of ARA-C- resistant 
cells by CCNU, which thus demonstrates the effectiveness of combination chemotherapy 
for circumventing the problem of drug resistance. 



Clinical Trials 



Hertz, et al., in 1961 first demonstrated chemotherapeutic cure of human cancer 
through the treatment of choriocarcinoma of women. He used intensive intermittent 
courses of methotrexate and quantitatively estimated reduction of cancer-cell popula- 
tions by monitoring the chorionic gonadotropin levels. The use of an intermittent treat- 
ment schedule was stimulated by the work of Goldin (1956) which demonstrated the value 
of such a regimen in treating L.1210 murine leukemia. Certain principles derived from 
these studies were later used to aid in the design of intensive combination chemotherapy 
of childhood acute lymphocytic leukemia (Frei and Freireich, 1965). 

Accounts of the use of combinations of agents appeared in the literature soon after 
early reports that human cancer would respond to chemotherapy. However, the early 
uses of such combinations were, for the most part, empirical. As more studies were 
published, the evidence indicated that combination therapy, if properly employed, may 
provide more successful treatment regimens. Progress in combination chemotherapy 
for childhood leukemia began to occur more rapidly, when, by the early 1960's, more 
rational approaches were designed and implemented in clinical studies. 

The lack of adequate means for control of hemorrhage and infections which result 
from depression of platelets and leukocytes inhibited the progress of single and com- 
bination chemotherapy in the 1950's. By the early to middle 1960's, platelet replace- 
ment and more successful means of controlling infections allowed the use of more 
intensive combination regimens and was a significant factor in the advancement of com- 
bination chemotherapy (Zubrod, personal communication). 



1-5 

Frei, et al. , in 1961 reported on studies from Acute Leukemia Group B which 
utilized 6-MP and methotrexate to treat acute leukemia in both children and adults. 
Results of this study showed that in children there was a better remission rate for com- 
bination therapy using these drugs than for either drug alone. The median duration 
of complete remission was not different for combination as opposed to single-agent 
therapy, but long-lasting remissions were more common with combination therapy. It 
was also shown that prior treatment with one of these antimetabolites had no adverse 
affect on later therapy with the other; therefore, these agents could be used successfully 
in sequence. 

Combination chemotherapy was employed by Zuelzer (1964) and Brubaker (1963) 
for maintenance of remission in ALL. The use of a cyclic regimen of 6-me rcaptopurine 
and methotrexate alternated at 3-month intervals increased the median remission 
period to about 12 months. 

Burchenal in 1953 recognized that continued treatment was necessary after remis- 
sions were induced in leukemia in order to prevent early relapse. It then became 
evident that antileukemic drugs have to be considered in terms of their ability to both 
induce and maintain remissions. Of the earlier chemotherapy agents, prednisone and 
vincristine were considered to be the most effective drugs for remission induction 
(Goldin, et al., 1971). In a series of studies, the average complete remission rate 
for prednisone in children was 48 percent (Bernard, et al. , 1968; Freireich, et al., 
1963; Vietti, et al. , 1965; and Wolff, et al. , 1967). Studies on the effectiveness of 
vincristine in the treatment of ALL in children (Evans, et al. , i963; Karon, 1962; 
Karon, 1963; and Selawry, 1963) showed an average complete remission rate of approx- 
imately 45 to 50 percent. 

Combinations of chemotherapeutic agents were shown, in several studies, to 
improve remission induction in childhood ALL (Frei, et al. , 1961; Freireich, et al. , 
1963; Karon, 1963; Frei, et al. , 1963; and Selawry, 1964). Such studies demonstrated 
that combinations of effective agents interact with additive and perhaps synergistic 
effects and, further, that some agents when used in combination have independent or 
nonadditive toxicities and, therefore, can be used at full dose levels. The clinical 
basis for much of the progress in combination chemotherapy resulted from this rationale 
(Frei, 1972). 

In the earlier trials, the more effective two-drug combinations for remission in- 
duction in ALL of children appeared to be prednisone plus vincristine (George, et al. , 
1966; Selawry and James, 1965), and prednisone plus 6-MP (Krivit, et al., 1966). 
Freireich, et al. , in 1963 initiated a four-drug treatment regimen for childhood acute 
lymphoblastic leukemia which included vincristine, 6-MP, prednisone, and metho- 
trexate (VAMP). This was the first treatment program designed specifically for the 
cellular cure of this disease based on the concept that multiple drug combinations may 
be capable of destroying all or nearly all leukemic cells. This program of therapy did 
provide significantly longer periods of remission than provided by single agents or 
other combination regimens (Frei, 1965). The same four drugs as used in the VAMP 
program, when combined in different dosage and treatment schedules (POMP), resulted 
in substantially longer periods of remission (Henderson, 1967). Treatments in this 
regimen were continued for 14 months and with the greater success in remission main- 
tenance, focused attention on the need for more prolonged therapy during remission. 

Frei and Freireich (1965), using the L1210 model for estimating total leukemic- 
ceU kill, made estimates of the number of leukemic cells prior to treatment and at 
time of relapse. Using these estimates, they calculated the percentage reduction in 



1-6 

the leukemic- cell populations as a result of treatment regimens using the VAMP pro- 
gram. These calculations, although estimates, indicated that very substantial reduc- 
tions in the total leukemic-cell populations were accomplished with this treatment 
regimen. This provided thrust to the use of intensified combination chemotherapy- 
directed at total eradication of leukemic-cell populations. 

"Total therapy" studies were begun in 1962 (George, 1968; Pinkel, 1971) and con- 
tinued in subsequent years (Pinkel, 1971; Aur, 1971; Aur, 1972). They were based on 
the principle of administration of all effective antileukemic agents and also largely on 
clinical studies of leukemic children rather than on laboratory models. The principal 
features of these studies included administration of combination chemotherapy over 
prolonged periods and prophylactic CNS irradiation with or without intrathecal metho- 
trexate. These studies demonstrated extended duration of remission and prolonged 
survival time by using four or more drugs for remission maintenance (George, 1968). 
These studies also showed the value of 2400 rads of cranial or craniospinal irradiation 
in preventing relapse due to CNS leukemia and therefore in prolonging complete remis- 
sion (Aur, 1971; Aur, et al. , 1972). These studies were of great significance since 
they resulted in greatly extended periods of remission, primarily through prevention 
of CNS leukemia. 

The point in time at which a philosophy of therapy gains clinical acceptance is a 
rather arbitrary judgment. However, by 1970, the majority of published clinical 
trials and certainly those with the greatest success in inducing and maintaining remis- 
sions in childhood acute leukemia involved some form of combination chemotherapy. 
Therefore, it is at this point that the accounts of historical events for this study were 
concluded. 



HISTORY OF THE CANCER CHEMOTHERAPY PROGRAM 
OF THE NATIONAL CANCER INSTITUTE 



The first international collaborative chemotherapy program sponsored by The 
National Cancer Institute was described in a publication by Shear, et al. , in 1947. 
There were only a few institutes in the forties in which both laboratory and clinical 
facilities were available, and this limited the application of laboratory findings by 
early academic investigators to clinical studies. The Sloan-Kettering Institute under 
Rhoads became the early leader in cancer chemotherapy throughout the world. Rhoads 
stimulated the interest of many chemical and pharmaceutical companies to submit 
materials to the early screening program established at Sloan-Kettering. By 1955, 
20, 000 chemicals and numerous fermentation products had been received for screening 
for anticancer properties. Groups funded by the National Cancer Institute and Ameri- 
can Cancer Society to prepare new agents often found that there were not sufficient 
numbers of screening facilities to evaluate their compounds. Efforts were made by 
N.C.I, to force these groups to find their own sources for screening as a contingency 
of grant funding. This resulted in delays in getting new drugs into the clinics for testing 

In December, 1952, a special meeting was convened by the National Advisory 
Cancer Council to discuss problems relating to clinical chemotherapy of cancer and 
probably represented a key event in triggering the development of a programmed effort 
in chemotherapy. Establishment of the National Advisory Cancer Council's Chemo- 
therapy Committee was an outgrowth of this meeting. 



1-7 

In 1955, the Cancer Chemotherapy National Service Center (CCNSC) was estab- 
lished. The major objectives of this program were to organize drug-development 
activity, evaluate drugs for anticancer activity in animals, perform preclinical studies, 
and establish clinical evaluations for promising drugs. Advisory panels were estab- 
lished and were intimately involved in establishing programs to provide support in 
procurement and evaluation of drugs to laboratories and clinics. 

Problems in program and resource development during the years 1956-58 were 
experienced by the CCNSC. Lack of proper delegation of authority to negotiate con- 
tracts despite Congressional authorization for fund allocation resulted in delays. It 
took more than 2 years for N.I.H. to obtain appropriate delegation of authority to 
negotiate costr reimbursement contracts. 

During the next few years, there were significant increases in the level of Con- 
gressional funding and therefore in programs for screening agents, and also in other 
experimental activities such as pharmacology, biochemistry, and mechanisms of drug 
action and resistance as applied to cancer chemotherapy. 

In a report to the National Advisory Council published in 1959, Gellhorn stated 
that efforts in the screening program had not led to the discovery of clinically impor- 
tant classes of compounds which were not already known and adequately exploited. He 
also criticized the clinical group studies which he felt embodied empiricism and repeti- 
tion which did not promote optimal progress. As a result of this report, it became 
evident that a time lag of 4 years existed between the accession of a chemical and its 
introduction to clinical trial, with several more years necessary for clinical evaluation. 
This is, however, similar to the time required by pharmaceutical firms to prepare a 
drug for marketing. 

During the years 1960-1965, programs began to develop along target and basic 
research lines. Rapid advances in management of acute leukemia in children by com- 
bination chemotherapy and rational approaches to dosage schedules suggested the 
feasibility of more directed and consolidated efforts for specific disease categories. 
In order to accomplish this for acute leukemia, the Acute Leukemia Task Force was 
organized in 1962 under the leadership of Zubrod. This resulted in aggressive pursuit 
by all the cooperative clinical groups of new leads in the treatment of acute leukemia. 
In 1965, clinical pharmacology centers were established. These centers include 
clinical pharmacologic, biochemical, and experimental therapeutics for optimal imple- 
mentation and clinical usage of new drugs for chemotherapy. 

The previous section was prepared in large part from a publication prepared by 
Zubrod, et al. , in 1966. This historical account of organized chemotherapy programs, 
although not specific for acute childhood leukemia, bears heavily on the progress made 
in developing both single-agent and combination chemotherapy for this disease. 



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I-IO 

Skipper, H. E., Personal Communication ( 1975) . 

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N. Y. Acad. Sci. , j_0, 82-83 (1948). 

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and Suguira, K., Ann. N. Y. Acad. Sci., _52, 1360-1378(1960). 

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(1967). 

Zubrod, G. C, in Management of the Patient with Cancer, T. F. Nealon, Jr., (ed.), 
W. B. Saunders Co., 249-265, Philadelphia, Pa. (1965). 

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Baker, C. G. , Cancer Chem. Rept. , _50, 349(1966). 

Zuelzer, W. W., Blood, 24, 477-494(1964). 



FIRST APPLICATIONS OF 
CHEMOTHERAPY FOR HUMAN CANCER 



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J-1 

APPENDIX J. THE CARDIAC PACEMAKER 
INTRODUCTION AND SUMMARY 



The totally implantable cardiac pacemaker represents one of the genuine triumphs 
of modern medical research and development. It is estimated that at the present time 
140, 000 implants of this device are now in operation throughout the world. The success 
of the pacemaker is impressive not only in the number of lives that are saved each year, 
but also because nearly all of the pacemaker patients can return to productive, com- 
fortable lives. The widespread use of the pacemaker can be attributed to two main 
reasons. First, it provides an extremely effective means of treatment of Stokes -Adams 
syndrome. Patients with implanted pacemakers return to normal activity without the 
burden of routine maintenance. The device is completely contained internally, and the 
absence of external connections enables the patient to move freely in his environraent 
with virtually no reminder that his heart rate is being regulated by a prosthetic device. 
The second reason for the widespread acceptance of the pacemaker is the high relia- 
bility of the device. This, in essence, results from the advances in solid-state elec- 
tronics and materials science that have occurred in the last 25 years. Ironically, as 
will be discussed later, the potential for the current widespread use of the pacemaker 
was not recognized by many of the principals who were involved in its development. 

The successful application of a completely implantable cardiac pacemaker resulted 
from the convergence of several scientific disciplines and technological developments. 
Essential to the operation of a pacemaker are reliable, miniaturized circuitry; a reli- 
able, long-term power source; and materials that are compatible with the body environ- 
ment. From a physiological standpoint, the successful operation of an artificial pace- 
maker is based upon an understanding of the basic bioelectric mechanisms involved in 
the generation of a cardiac contraction. Thbs involves a knowledge of the conduction 
pathways in the heart, the polarization of the cardiac cells, membranes, and the re- 
sponse of the cells over the time frame of the cell membrane polarization cycle. From 
a clinical perspective, the operation of a pacemaker is dependent upon proper surgical 
and manipulative procedures for implantation of the device and insertion of the elec- 
trode(s) into or onto the heart. These technological developments and basic insights 
were brought together in 1958 with the first human implant of an entirely self-contained 
internal pacemaker. Since that time, a great many improvements have been made in 
power sources, electrodes, circuit reliability, versatility, and methods of implantation. 
The record of the ability of pacemaker devices to treat heart block effectively is indeed 
impressive. As mentioned above, approximately 140, 000 devices have been implanted 
since 1958 and approximately 30, 000 currently are implanted each year in the United 
States. The life expectancy of an artifically paced patient is over 90 percent of that of 
the normal population of comparable age and sex. The mortality rate in unpaced 
patients with severe heart block is about 50 percent over a 1-year period, so the effects 
of the pacer are quite dramatic. However, one should not infer from the performance 
record of artificial pacemakers that all the problems are solved. Problems still exist 
because of the limited lifetime of the power source, the development of high threshold 
potentials, and the occasional fracture of lead wires. Research on several approaches 
to these problems is under way. With respect to power sources, these developments 
include nuclear power sources, lithium iodide solid-state batteries, and various bio- 
electric power sources. New electrode configurations and methods of attachment are 
being developed to address the problem of high threshold potentials. These develop- 
ments will very likely lead in the near future to optimized pacemaker systems which 
will perform in a trouble-free manner without any manipulation over a 5- and possibly 
10-year period. 



J-2 

The basic pathological condition which is corrected by the artificial pacemaker is 
commonly referred to as heart block or Stokes -Adams syndrome. The stimulus for the 
contraction of the heart is bioelectric in nature and arises in the sino-atrial (S-A) node 
of the atria. Transfer of the stimulus to the ventricles occurs through the atrio- 
ventricular (A-V) conduction system which consists of the atrioventricular node and 
the atrioventricular conduction bundle. In A-V heart block, the sino-atrial stimulus 
is not conducted to the ventricles so they do not contract rapidly and regularly. The 
artificial pacemaker delivers a small electrical impulse to the ventricles, causing them 
to contract, and thus serves as a substitute for the natural pacemaker system. 

The history of the development of the cardiac pacemaker provides a fascinating 
study of the impediments which can prevent a potentially beneficial method of treatment 
from reaching the patient. The invention of a pacing device seems to have recurred 
several times in the last hundred years, each time only to be discarded because of the 
absence of sufficient supportive technology, unfavorable response in the scientific com- 
munity, or a lack of understanding of the implications of the results which were 
obtained. In 1791, Galvani made the observation that the heart of a frog could be stim- 
ulated by an electrical impulse. Subsequent to this, various demonstrations on the 
human heart were made in the late 18th and early 19th centuries. Duchenne de Bologne 
suggested in 1892 that electrical stimulation would be an effective method of treating 
cardiac arrest. Some 65 publications on the electrical stimulation of the heart had 
appeared by 1932. At that time, the work of Hyman laid the definitive foundation for 
the development of the pacemaker. Hyman fabricated a device which delivered a timed 
series of electrical pulses to the heart by means of a bipolar needle electrode which 
was inserted through the chest wall into the right atrium. He termed this device the 
"artificial pacemaker", and demonstrated its efficacy for treatment of cardiac arrest 
in several human patients. The work of Hyman generally was not well received by his 
medical colleagues, or, for that matter, by the public in general. In fact, Hyman did 
not publish his human studies even though he alluded to them in his publications. He 
was subjected to several lawsuits because of application of the pacemaker. The only 
manufacturer willing to produce the device was in Germany, but by the time prelimi- 
nary arrangements were made. World War II had begun, and the device was never 
manufactured. Because of these impediments, the concept of the artificial pacemaker 
lay fallow for the next 20 years. Interest was reawakened in 1952 by the work of ZoU 
and his colleagues who applied various external electrodes on the chest wall and were 
successful in treatment of patients with heart block. The external electrodes also 
stimulated skeletal muscles and in some cases caused skin irritation, and thus were 
unsuitable for chronic stimulation. With the impetus provided by ZoU's work, devel- 
opments occurred rapidly in the next several years. Lillehei and co-workers devel- 
oped a system with internal electrodes and a portable external pacer. The first com- 
pletely implantable artificial pacemaker was implanted in a human patient in 1958 by 
Elmquist and Senning. The device consisted of a miniaturized transistor pacemaker 
which was implanted in a subcutaneous epigastric pouch and electrodes which were 
placed in the heart. Ironically, Senning became discouraged with the device because 
of the continually rising potential needed to stimulate the heart, and in his first report, 
he stated that another approach for treating A-V block was needed. 

The development by Hunter and Roth in 1959 of a bipolar electrode which main- 
tained a stable threshold potential paved the way for successful long-term implants. 
This was finally achieved in I960 by Chardack and Greatbatch who designed and im- 
planted a pacemaker which incorporated the Hunter-Roth electrode and a self-contained 
mercury battery. 



ll 



J-3 
HISTORICAL ACCOUNT 



The marvelous stimulatory effect of electric current on skeletal muscle and 
various other body functions was a well-documented phenomenon in the latter portion 
of the 18th century. Galvani's work in 1791, in which he applied electric current to 
nerves, muscles, and hearts of dead frogs, was well known. He found that the skeletal 
muscles could be induced to contract by the application of galvanic current and that the 
susceptibility of the muscles to stimulation gradually decreased over a period of time. 
The heart was first to lose its ability to be stimulated. Ironically, the effects of elec- 
tric current on human subjects were noted many years before the experiments of 
Galvani. Probably the first physiologic effect of such currents was noted in 1744 by 
Kratzenstein who observed that electric currents caused a mild elevation in pulse rate. 
Subsequent to this, many investigators verified that electric current did indeed produce 
a mild tachycardia in human subjects. A remarkable account of the use of electric cur- 
rents to stimulate the heart in cardiac arrest was written by Aldini (1804) who described 
the successful resuscitation of a 3-year-old child who had fallen down stairs. Electric 
pulses were applied to various portions of the child's body without success until the 
electrodes were placed across the thorax. This resulted in a pulsation, and, according 
to the report, the child recovered completely. The success of this one case, however, 
was masked by failures by many other investigators to reproduce these results. Never- 
theless, studies of the effect of electric current stimulation continued. Bichat (1800) 
experimented with bodies of guillotined criminals 3 or 40 minutes after execution. He 
was able to stimulate pulsations of the heart only by direct contact of the surface of the 
heart with electrodes. The work of Bichat and others was summarized in 1804 by 
AlH'.ni. In Aldini's further experiments with executed criminals, he showed that the 
heart lost its ability to be stimulated by electric current more rapidly than other 
muscles. Work on the electrical stimulation of the heart continued throughout the 19th 
century. For example, in 1824, Reese described a method for reanimation of a patient 
with cardiac arrest which consisted of passing one electrode down the esophagus and 
attaching another one to various parts of the external surface of the body. In the mid- 
19th century, Duchenne experimented extensively with various forms of electrical stim- 
ulation to treat a wide variety of cardiocirculatory ailments. He advocated treating 
angina pectoris with a very strong faradaic current passed through the left nipple. He 
related that the terrible pain produced by the current caused the angina to disappear. 
His work extended for many years, and, in 1872, he described an electrical method for 
stimulating the heart during cardiac arrest. In this method, one electrode was placed 
somewhere on the skin of the patient. The other electrode was held in the clinician's 
hand and was tapped rhythmically on the precordial area of the patient. This was said 
to stimulate the heart rhythmically and Duchenne claims to have successively stimulated 
the heart of a patient suffering with bradycardia. 

In 1871, the work of Bowditch laid the foundation for one of the important con- 
cepts of present-day cardiac potential — the stimulation threshold. Bowditch showed 
that a certain potential was necessary to stimulate the heart. Above this potential, 
there was no increased strength of the contraction. Below the potential, no stimulation 
occurred. Further basis for present-day pacing technology was established by von 
Ziemssen in 1882 who found he could accelerate the heart rate in a human subject by 
interrupting the applied current at a rate faster than the natural rate. Further defini- 
tive work on the effect of galvanic currents on the heart was described in 1889 by 
MacWilliam. He verified earlier work on the acceleration of a beating heart by direct 
application of electric current to the heart itself. He also found that too great a cur- 
rent could cause fibrillation, and that a quiescent heart could be excited to a rhythmic 
series of beats by the application of an appropriate level of current. 



J-4 

An important milestone in understanding the physiology of the beating heart re- 
sulted from the work of His in the 1890's. He showed the propagation of the ventricular 
contractile stimulant through the atrioventricular bundle (now known as the bundle of 
His) by cutting through the bundle in rabbit hearts (His, 1893). 

The use of an intracardiac electrode to stimulate the heart, which was developed 
in the 1950's, was predated by over 50 years by the work in 1899 of Chaveau who in- 
serted a catheter into the left ventricle of the heart. This catheter was designed in such 
a way that a small electrogalvanic impulse was generated when the aortic valve closed. 
This caused a great acceleration of the heart and resulted in the death of the animal. 
Further work on an endocardial catheter was performed in 1905 by Floresco who in- 
serted a glass rod through the external jugular vein into the right ventricle. The glass 
tube contained copper or platinum wires and was capable of stimulating the heart. The 
dogs used by Floresco were poisoned with ether and then resuscitated by electro- 
stimulation. 

At the beginning of the 20th century, much of the basic groundwork had already 
been laid for the work that was to follow in successful application of the pacemaker. 
Unfortunately, much of the significance of this earlier work was obscured by the 
plethora of electrical devices and applications which \^fere being promoted in the early 
1900's to cure almost every disease and ailment of the human body. A great many ail- 
ments of the cardiovascular system from angina pectoris to congestive heart failure 
were treated by a wide variety of electrostimulation methods. Stimulation was achieved 
by methods including stimulation of the nerves of the neck, application of external elec- 
trodes on various parts of the body, and internalized needle electrodes. A host of dif- 
ferent devices was employed, and often extravagant claims were made about the efficacy 
of the treatments. Patients were immersed in electrified bathtubs, placed in solenoid 
cages, or laid on electrified couches. Often the amount of current utilized was governed 
by how much the patient could stand. These abuses of electrotherapy resulted in severe 
misgivings among much of the medical community about safety, efficacy, and discom- 
fort to the patient. As a result, serious consideration of electrical stimulation was at 
a very low level for the first 20 years of this century. 

The modern basis for pacemaker therapy was inaugurated by the work of Hyman 
starting in 1928. Dr. Hyman had been interested in the problem of stimulating the 
arrested heart for a number of years. In 1926 with the establishment of the Witkin 
Foundation at Beth David Hospital in New York, he was given the opportunity of investi- 
gating therapeutic methods of cardiac resuscitation. At that time, intracardiac injec- 
tions were used by some clinicians for stimulating the heart in cardiac arrest. Hyman 
noted that a large number of different drugs were utilized in the intracardiac injections 
and the results did not seem to depend upon the type of drug used. He therefore rea- 
soned that the important factor of these injections was not the pharmacological action of 
the drug itself but the action current of injury resulting from the puncture wound of the 
needle in the cardiac tissue. He further concluded that a more reliable method of stim- 
ulating the heart would be to provide a small electrical impulse which simulated the 
normal sinus pacemaker until such time as it could resunne its normal activity. He was 
reinforced in his conclusion by much of the background work done in the previous 50 
years. 

In 1929, Hyman experimented with a number of laboratory animals to define the 
conditions under which his artificial pacemaker could regularly stimulate the heart. In 
1930, he received a grant from the Witkin Foundation which enabled him to develop the 
first practical artificial pacemaker. This device had several of the basic elements of 
the currently used pacemaker system. These included a power source, a method of 



J-5 

interrupting current, a timing device, and an electrode for delivering the stimulus to 
the heart. The device that Hyman produced, which was the first artificial pacemaker, 
weighed about 15 pounds. 

Hyman's device consisted of a magnetogenerator for the power supply. A spring- 
wound motor with a ballistic governor was used to drive the magnetogenerator. The 
rate and duration of the pulses were controlled by a rotating two-phase interruptor disk. 
With one winding, the device could maintain pacing for a 6 -minute period. This, of 
course, meant that the device had to be wound every 6 minutes, but since it was not 
originally intended for long-term therapy, this feature was not a severe drawback. 
The output electrodes of the device were connected to a bipolar needle which was in- 
serted through the chest wall into the right atrium. 

Hyman experimented with this device on animals and was convinced of its effec- 
tiveness by the end of 1931. The device was then applied to human patients, and by 
March, 1932, the device had been used in 43 patients with a successful outcome in 14 
cases. Hyman published his results in 1932 and described his device and results in 
animals. He did not publish the results of his clinical studies in this paper, but indi- 
cated at the end of the paper that he felt it was safe enough to be used in human subjects. 
A possible reason why he did not publish the clinical results was a growing opposition to 
his work in the medical and lay community. In an attempt to promote the concept, 
Hyman undertook a speaking tour, but this appeared to polarize the advocates and the 
detractors to this technique even more. Many people regarded his endeavors in cardiac 
resuscitation as irresponsible tampering with Divine Providence. So strong was the 
objection to his device that he was named the defendant in lawsuits (eventually unsuccess- 
ful) totalling over $11 million because of his use of this device. His difficulties in 
obtaining acceptance were further compounded by the fact that he was unable to find any 
American manufacturer who was willing to produce the device. He did succeed in 
reaching an agreement with Siemens Corporation in Germany at the end of the 1930's. 
World War II interrupted this association, and after the war Hyman did not continue to 
pursue pacemaker development. 

A more sophisticated understanding of the basic requirements for cardiac pacing 
resulted from the work of Wiggers and Wegira in 1940. These workers defined periods 
in the cardiac cycle when the heart could be fibrillated and also determined the nature 
of the electric current that could terminate the fibrillation. With respect to pacemak- 
ing, they studied the effects of electric stimuli of varying intensity, interval, timie, and 
duration on pacing the heart. They further demonstrated the vulnerable phase in the 
cardiac cycle, a phase in the relative refractory period when a strong stimulus pro- 
duces multiple beats and/or fibrillation. The first clinical use of direct shock to the 
heart to counteract fibrillation was performed in 1946 by Beck, Pritchard, and Fell. 

After the war, interest was again renewed in cardiac pacing. The tremendous 
developments in electronics which occurred during the I940's provided a technical stim- 
ulus to the development of a practical pacemaker. Another stimulating factor for the 
reawakened interest in pacemaking was the rapid development in intracardiac surgery. 
In the late 1940's, the laboratory headed by Wilfred Bigelow at the Banting Institute of 
the University of Toronto developed a technique of hypothermia for cardiac surgery. 
Because cardiac arrest occurred at temperatures lower than 20 C, this group was in- 
terested in methods of stimulating the heart after surgery. They developed an elec- 
tronic pacemaker which delivered impulses by means of an intracardiac bipolar elec- 
trode which was placed in the right atrium. In experimental work with dogs, they were 
able to show that the pacemaker could reestablish beating of the heart in animals cooled 



J-6 

to below 20 C. They also showed that the pacemaker could control the rate of a heart in 
a dog at normal temperatures. Because the popularity of hypothermia for intracardiac 
surgery was on the decline at that time, the device did not find clinical use in hypo- 
thermia patients. However, the device was used unsuccessfully in five patients with 
cardiac arrest. In these patients, the electrode was placed in the right atrium by the 
transvenous route. Dr. Callaghan (cited in Schechter, 1972) noted that had they pushed 
the catheter 2 inches farther down into the right ventricle, the device might well have 
succeeded in resuscitating the heart and inaugurating the era of cardiac pacing 10 years 
earlier than it had occurred. 

The work of Bigelow, et al. , did provide a stimulus for further developments of 
the pacemaker. In 1951, Dr. Paul ZoU contacted them and requested information on 
the design of their pacemaker. ZoU had been contemplating the use of electrical stim- 
ulation for cardiac arrest for some time. His objective was not merely the initial 
stimulation of an arrested heart but the use of a pacemaker for treatment of Stokes - 
Adams syndrome. Accordingly, he reasoned that an atrial pacemaker would be in- 
effective in cases where A-V block had occurred. He therefore felt that the needle elec- 
trodes of Hyman and the transvenous electrode of Bigelow, et al. , should be replaced by 
a different system of electrodes. At first, he advocated placing one electrode in the 
esophagus under the heart and the other electrode in the precordial area over the heart. 
In later experiments with dogs, he found that equally effective stimulation could be ob- 
tained by electrodes placed externally on the chest wall. The first clinical application 
of the external pacemaker was reported in 1952. In essence, the technique was effective 
in resuscitating the heart in cardiac arrest but proved unsatisfactory for long-term 
stimulation in heart block patients because of discomfort resulting from the external 
electrodes. Nevertheless, the work of ZoU is significant because it demonstrated the 
efficacy of a pacemaker system in reliably establishing the heart rate over a period of 
time. 

In the mid-1950's with the great increase in the clinical application of open-heart 
surgery, a new need for cardiac stimulation resulted from iatrogenic heart block. 
Responding to this need was a group headed by C. Walton Lillehei at the University of 
Minnesota who developed a portable electronic stimulator. The pacemaker was con- 
structed with transistors and was powered by a mercury battery. In the first clinical 
applications of the device in 1957 (Weirich, Gott, and Lillehei, 1957), one electrode 
from the pacemaker was anchored into the right ventricular wall and the other electrode 
attached externally on the skin. After the first few cases, the indifferent electrode was 
implanted subcutaneous ly over the apex of the heart. The use of the device was later 
extended to cases in which thoracotomy was not performed. In this case, the stimula- 
tion needle was introduced transcutaneous ly through a needle. By the end of 1958, this 
group had used the pacemaker on 72 patients (Weirich, et al. , 1958). The device was 
portable and allowed the patients to be ambulatory. The longest pacing lasted almost 
2 months. 

These developments showed the feasibility of electrical stimulation of the heart 
for a variety of conditions. One of the drawbacks in the late 1950's toward further clin- 
ical use was the electrode system that delivered the stimulus to the heart. Numerous 
problems were encountered with a buildup in the threshold potentials with monopolar 
electrodes planted in the heart wall. In 1958, a large step forward in electrode devel- 
opment was made by Furman and Schwedel who placed a unipolar electrode in the right 
ventricle. This system was introduced by a transvenous route and consisted of an ex- 
ternal pacemaker and an indifferent electrode implanted subcutaneous ly on the chest 
wall. In the first human patient in which this system was used, pacing was maintained 



J-7 

for 96 days. The patient, who had been suffering from recurrent Stokes-Adams syn- 
drome, survived with no untoward reactions. This demonstration of the efficacy of a 
transvenous application in the right ventricle paved the way for more recent develop- 
ments in bipolar ventricular electrodes. At the present tirae, about 90 percent of 
implantable pacemakers utilize electrodes delivered transvenously. 

With the demonstration that pacing could be continued for relatively long periods 
of time, the undesirability of an external pace generator became more evident. 

Even though the use of transistorized pacemaker circuitry and efficient power 
sources enabled the miniaturization of the device, the necessity for percutaneous leads 
carried the potential for infection. The fact that the device was external was a constant 
reminder of its presence to the patient. The next logical step in the development of the 
pacemaker was a completely self-contained system in which the generator and the elec- 
trodes were contained inside the body. In 1958, Elmquist and Senning developed such a 
device which was designed to be implanted in an epigastric pouch. The system used a 
rechargeable nickel-cadmium battery which could be recharged by inductive coupling 
from outside the skin. In October, 1958, this device was implanted in the first human 
patient. Unfortunately, the threshold potentials for this patient continued to rise and 
after approximately 5 weeks had risen to 20 volts. In the first report on this device 
(A. Senning, 1959), Dr. Senning was discouraged by the high threshold potential and felt 
that another way must be developed to treat chronic A-V block. The problem encoun- 
tered by Dr. Senning did not occur because of some basic flaw in the concept of pacing 
or in the pace generator but because of an improper interface with the electrode to the 
myocardial wall. 

In 1959, Hunter and Roth developed a bipolar electrode which went a long way 
toward solving the problem of the electrode interface. This electrode consisted of two 
stainless steel spikes mounted on a silicone rubber platform. The unit was sutured 
onto the myocardial wall with the two electrodes buried in the myocardium. This elec- 
trode was implanted in a human patient in 1959 using an external pacing unit. The 
patient subsequently refused to have an internal generating system implanted after they 
were available. 

The culmination of all of the advances in pacemaker technology which occurred in 
the 50's was realized in the work of Chardack and Greatbatch. They developed a pace- 
maker system which incorporated the Hunter -Roth electrode and was powered by self- 
contained mercury cell batteries (Chardack, et al. , I960). The device was 6 cm in 
diameter and 1. 5 cm thick. The generating circuit was transistorized and contained 
only 8 components. The output of the device was a square pulse of about 10 volts 
amplitude and 1 msec duration delivered at 60 pulses per minute. Because of the 
efficiency of the Hunter-Roth electrode, the required power output was very small and 
the batteries were capable of powering the unit for a relatively long period of time. 
The first device was implanted in April, I960, and the device performed very well 
(Chardack, et al. , I96O). This device with few modifications was essentially the sarae 
design that was subsequently marketed by Medtronic and initiated the large clinical 
acceptance of pacemaker devices. 

The historical account of pacemaker development has followed the chain of events 
relating to the development of the device and its application in human patients. It is 
important to emphasize that the successful development of the pacemaker could not 
have been accomplished without the availability of several technological innovations 
which occurred in the period from 1940 to I960. These innovations included the 



J-8 

transistor, the development of reliable batteries for power supply, and the development 
of biocompatible materials used in the construction of the pacemaker. 

Semiconductor electronics were essential in the totally implanted pacemaker. 
The small size, high reliability, and low power requirements which enabled the long- 
term implantation of the pacemaker would not be possible with vacuum tube electronics. 
It is beyond the scope of this study to trace the basic work in electronics and solid- 
state physics which led to the development of the transistor. Suffice it to say that the 
key events in this development were initiated by the quantum mechanical model of solid 
semiconductors. A patent on silicon P-junctions was obtained in 1941 by R. S, Ohl, and 
this development found extensive use during World War II in radar applications. After 
the war, research and development on semiconductors continued at an increasing pace. 
In 1948, Bardeen and Brattain's publication on the transistor opened the door for the 
era of solid-state electronics. 

Battery technology played an extremely important role in the implantable pace- 
maker because a long-term, reliable power supply is absolutely essential. The typical 
power output from a pacemaker ranges from between 50 and 70 microwatts. With a 
50 percent efficiency, the average battery drain is in the range of 200 milliampere- 
hours per year. For a reasonable battery year, then, a capacity of about 1 ampere- 
hour is required. The batteries cannot produce gases in their operation since they 
must be placed in a sealed unit. The mercury/zinc cell utilized by Greatbatch and 
Chardack meets this requirement and its development is traced in the next few 
paragraphs. 

The forerunner of the dry cell was developed in 1868 by Leclanche. This cell 
uses a paste or gel electrode and a depolarizer to minimize gas buildup. In 1884 Clark 
patented a cell which used mercuric oxide as a cathodic reactant. The need for reliable 
dry cells became critical in World War II and this stimulated research in this area. In 
1947, Ruben patented a zinc/potassium hydroxide/mercuric oxide dry cell which he had 
developed during the war. He worked in conjunction with the P. R. Mallory Company, 
and the cell is referred to as the RM Cell, a contraction of both names. The cell has 
the desirable properties of minimal gas evolution, long life, and a flat discharge curve. 
The battery was improved over the years, and by 1959, it was suitable for encapsula- 
tion in epoxy resins and use in the implantable artificial pacemaker. 

In the area of materials, two types of synthetic polymers were also essential for 
the successful implantation of the pacemaker. The first of these was silicone rubber. 
Long-term implantation of the electronics in the body requires that it be covered by a 
material which is compatible with the body tissues and which is resistant to degradation 
in the body environment. Silicone rubber (or polydimethylsiloxane) is such a material 
and has been used extensively in in vivo applications. This type of polymer was dis- 
covered accidentally in 1905 by Kipping in his attempt to make a silicon analog of a ke- 
tone. At the time, the material was not considered useful and the first industrial appli- 
cation of these polymers was not until the 1930's at Corning Glass Works. At that time, 
Hyde did extensive work on synthesizing and characterizing silicone polymers. In the 
late 1930's, Corning Glass and Dow Chemical Company combined efforts to continue 
development of silicone, and by 1943, the first pilot plant for silicone production was 
built by Dow Corning. In 1944, Rochow at General Electric outlined a new technique 
for the facile synthesis of the precursor dimethyldichlorosilane from silicone and 
methyl chloride. Because of the inert nature of the silicone polymers, Dow Corning 
began to explore their potential application in the field of medicine. Medical-grade 
silicone rubbers were first produced in 1953 by Dow, and in 1959, the company 



J-9 

established the Dow Corning Center for Aid to Medical Research. The performance 
and availability of silicone rubbers thus made it the logical choice for encapsulation of 
the pacemaker. 

Another important synthetic polymeric component of the pacemaker is the epoxy 
potting compound in which the batteries and electronics of the pacemaker are encapsu- 
lated. The groundwork for modern epoxies was laid by Paul Schlack in a German patent 
which described various evaporation techniques and a hardening process for liquid 
polyepoxides. One of the pioneers in the development of epoxies in the l940's was a 
Swiss scientist, Pierre Castan, who obtained a patent in 1943 covering the curing of 
phenolic epoxide resins with dibasic acids. In the early 1950's, Greenlee and his 
associates at DeVoe and Reynolds advanced the knowledge of the chemistry and potential 
applications of epoxy resins. At the same time, workers at Shell Development obtained 
a series of patents for the uses of epoxies as adhesives, coatings, and potting com- 
pounds. The potting compoiond used by Greatbatch was produced by the Minnesota 
Mining and Manufacturing Company. 

Exclusive rights to the pacemaker developed by Chardack and Greatbatch were 
obtained by Medtronic, Inc. , who saw a considerable potential for the device. Their 
predictions turned out to be true, and in the early 1960's, the widespread clinical appli- 
cation of the device had indeed taken place. Although the historical narrative of the 
development of the pacemaker does end at this point, it should be pointed out that many 
improvements on the original device have been made in the years from I96 to the 
present time. The Chardack-Greatbatch pacemaker was of a type designated as an 
asynchronous type; that is, it developed a stimulus at a constant rate between 60 and 
80 beats per minute. The rate of the pacemaker was fixed durings its manufacture. In 
1962, Kantrowitz and co-workers developed a pacer which allowed selection of the fixed 
rate to suit the needs of the patient. The method of asynchronous pacing is still widely 
used and meets the needs of a large number of patients. However, in the early 1960's, 
it was felt that certain types of patients needed a certain degree of physiological con- 
trol of the pacemaker. In 1963, Nathan and co-workers developed the synchronous 
pacemaker which, in essence, serves as a system to conduct the naturally occurring 
stimulus generated by the S-A node to the ventricles. A third type of pacemaker was 
developed in 1965 by Berkovits and associates. In this type of device, termed the de- 
mand pacemaker, the pacemaker delivers a stimulus to the heart only in the absence of 
a naturally occurring stimulus. The circuitry is so designed that the natural stimulus 
is sensed by the pacemaker from the R-wave of the electrocardiograph signal. 

Numerous other improvements have been made in electrode materials and 
designs. Finally, a considerable amount of effort has gone into development of various 
types of power sources which will deliver reliable power over a long period of time, 
preferably over 5 years. Greatbatch (1971) has developed a solid-state lithium iodide 
battery which has a projected lifetime of 10 years. Several different groups have been 
involved in the development of a nuclear-powered pacemaker energy source and clinical 
trials of this system have begun. In addition, numerous other approaches involving 
bioelectric fuel cells, piezoelectric systems, and biogalvanic cells have been devised 
for powering the pacemaker. It is likely that many of these developments will be uti- 
lized in the future to make the pacemaker even more reliable. 



J-10 

REFERENCES 

Aldini, L'unprimerie de Fournier fils, Paris (1804). 

Bardeen, J., and Brattain, W. H. , Phys. Rev., 73, 230 (1948). 

Beck, C. S. , Pritchard, W. H. , and Fell, H., JAMA, ^35, 985(1947). 

Bichat, X. , Recherces Physiologiques sur la "Vie et la Mort , Paris, Brosson, Bagon & 
Cie (1800). 

Bigelow, W. A., Callaghan, J. C. , and Hopps, J. A., Ann. Surg., 132 , 531 (1950). 

Bowditch, cited by Faivre, et al. , L'Entrainment Electrique der Coeur, Masson et 
Cie, Paris (1964). 

Castan, P., Swiss Patent 21 1, 1 16 (1940). 

Chardack, W. M. , Gage, A. A., and Greatbatch, W. , Surg., 48, 643 (I960). 

Chaveau, J., J. Physiol. Path., }_, 377 (1899). 

Clark, C. L. , U. S. Patent 298, 1 75 (1884). 

Duchenne, G. , Gag. Hop. (1853). 

Floresco, N. , J. Physiol. Path., 7, 785 (1905). 

Furman, S. , and Schwedel, B. , New Eng. J. Med., 26, 943 (1959). 

Galvani, L. , Bologna Inst. Sci. (1791). 

Greatbatch, W. , Lee, J. H. , Mathies, W. , Eldridge, M. , Marey, J. R. , and 
Schneider, A. A., IEEE Trans. Biomed. Eng., ^1, 1317 (1971). 

His, W., Arb. Med. Klin. Leipzig, 2, 141 (1893). 

Hunter, S. W. , Roth, N. A., Bernardez, D. , and Noble, J. L. , Lancet, 7^, 506 
(1959). ~" 

Hyman, A., Arch. Int. Med., 46, 553 (1930). 

Hyman, A., Arch. Int. Med. , _50, 283 (1932). 

Kantrowitz, A. , Cohen, R, , Raillard, H. , Schmidt, J. , and Feldman, D. S. , Surg. 
Gynec. Obstet. , n_5, 415 (1962). 

Kipping, F. S. , Proc. Roy. Soc. , A139 , 139 (1937). 

Leclanche, G. , cited by Vinal, G. W. , Primary Batteries , John Wiley & Sons. 

Lemberg, L. , Castellano, A., and Berkovitz, B. , JAMA, 191, 12 (1965). 



J-11 

MacWilliams, J. A., J. Physiol., _9, 167 (1888). 

Mamorstein, M. , J. Physiol. Path., 24, 40(1927). 

Nathan, D. A., Center, S. , Wu, C. Y. , and Keller, W. , Circ. , 27, 682 (1962). 

Ohl, R. S., U. S. Patent 2,402,662 (1941). 

Reece, R., Medical Guide for the Use of the Clergy, Hearts of Females and Junior 
Practitioners in Medicine and Surgery , 14th Ed. , London, Longman, Hurst, and Co. 
(1824). 

Rochow, E. G., cited by Burkhard, C. A., Rochow, E. G. , Booth, H. S. , and Hartt, 
J., Cham. Rev., 47, 97 (1947). 

Ruben, S. , U. S. Patent 2, 422, 045 (1947). 

Schecter, D. , N. Y. State J. Med., 605 (1972). 

Schlack, P., German Patent 676, 117 (1939). 

von Ziemssen, H. , Arch. Klin. Med., 30, 270 (1882). 

Weirich, W. L. , Gott, V. L., and LiUehei, C. W. , S. Forum, 8, 360 (1957). 

Wiggers, C. J., and Wegira, R., Amer. J. Physiol., 2_28, 500 (1940). 

ZoU, P. M. , New Engl. J. Med., 247, 768 (1952). 



BACKGROUND 

1 791 Gal\ 
1800 Bich 

1804 Aldi 
1824 Rees 

1871 Bow 

1872 Duel 

1882 Von 

1893 His I 
I 

1899 Chav 
1926 Dr 



BATTERY DEVELOPMENT 



BACKGROUND IN ELECTRICAL STIMULATION 



ia04 Aldint Account of (CiuscitaKon bv 

4-ye3r-o1d child 
1824 Reasa Method for ireating cardiac- 

arreit. Esophogeal and emein, 

1B7I Bomditcti Defined tllmulaiion Ihrei- 

hold 
1872 DucheiHio Summamod exiensive 



could b« eccslarated bv 
pultod eleciric current 



1926 Ourv Enpeiimenied «ith reltactoty 
period in frog hearts 

NR/S/U/P/M 



PACEMAKER DEVELOPMENT 



MR/D/U/P/M 

- 1 930 Hyman Developed lirsi protolypa 

device lor electrical cardiac 

MR/D/U/P/M 

- 1931 Hyman First clinical application o' 

pacornaker in 41 pallcnit 
MR/D/U/P/M 

- 1932- Hyman encounten appojiiion to pa 
1939 maker, ii unable lo lind ma> 



MR/S/ / / 

_1»40 Hyman WWII inlerrupti aiEOCiBI 

viith Siemens Corp. in Garmany. 

MR/S/ / / 

- 1940 Wiggers Defined periods in cardiac 

NR/S/U/P/M 

■ 1946 6eck First clinical use of direct shock 
to counteract librillalion 
MR/S/U/P/M 

-1950 SiQeloiiv Developed emernal pacemaker 

MR/D/ I? I 

- 1951 2oll Began ivork an entetnal pacemaker 

MR/D/U/G/M 

- 1952 Zoll Succeulully utad pacemaker o 

Pacing conlinued for 5 days 
MR/D/U/G/M 



'S and physicians 

NR/S/ / / 



1953 Callaghan Paoem 



MR/S/W/G/M 
■i Developed external paccnu 



- 1958 Welrich Successful clinical applicaiio 

Pacing lasted up to 2 month 
MR/S/U/P.G/M 

- 1953 Elmquisi Developed and used clinically 

completely implantable pam 
maker. Encountered high 
stimulating potential 
MR/S/W/G/M 
-1958 Furman Intracardiac electrode Intio- 
duced iniiavenously used 
clinirallv for liealmenl ol 
heart block 

MR/D/U/G/M 

-1959 Hunter Developed plallorm etectfOde 
which solved many problems 
of high stimulation potential 

MH/D/U/P/M,C 



MR,D/D/U/P/M,C 

1950 Medtronic Obtained rights to Grent- 
baich-Ch^rdack pacemaker 





MATERIALS DEVELOPMENT 


TRANSISTORS 


1868 LeClanche Developed cell kvhich was 

lorerunner of dry cell 




— 1905 Kipping Discovered silicone polymers 
accidentally 


1920 Development o( quantum mechanics 


— 1B84 Clark Incorporated mercuric oxide as 
calhodic reactant lor battery 


" 


NR/S/ / / 


1930 by Pauh, Fermi. Ditoc, Schroedingor, 
Heisenberg. provided baiKground for 

NR/S/ / / 






— 1 930 Hyde Did extensive developmental 








1935 i,vork on silicone polymers 








DV/S/U/P/C 


1931 Wilson Developed quantum mechanical 

model of semiconductor 
NR/S/W/G/M 






— 1939 Schlack Established background (or 








DV/S/W/P/C 








— 1940 Caitain Obtained U.S. and Swiss 






^' 


1943 patents on production and 








curing of epoxy resins 


1941 OhI Obtained patent on silicon p-n 






— 1943 Don Corning Corp. Firsi pilot plant 








lor silicone produc- 


MR/D/U/P/C 






DV/S/U/P/C 








— 1946 Rochow Method for facile synlhesij of 








monomers (or silicone polymers 


1948 Bardeen Si Invented transistor and 


having long life and minirrul ^i 




DV/S/U/P/C 


Braltain published first description 








MR/D/U/P/C 


DV/D/U/P/C 






1950 E.teniive oommarcial devalopmeol 


——1948 Rubin Continued refinomeni of 




1956 3-M Corporation 


1950 of (ransisto- technology 


1951 mercury cell. Sealed unit 




Oevoe & Reynolds Co. 

Commercial development 


DV/S/U/P/C 


DV/S/ / / 




of epoxy resins 






er 


DV/S/U/P/C 








1953 Dow Coming Corp. Produced first 








medical grade 








DV/S/U/P/C 












CODES 
NR Nonmission-Oriented Research 
MR Mission-Oriented Research 
DV Development 
S Significant Event 
D Decisive Event 
U U. S. Event 
W Foreign Event 
G Government Funding 
P Private Funding 
M Medical School - Hospital - 

Unive'siiy 
I Research Institution 
Gt. Government Laboratory 








C Company 



Many other advances end 1 


novatio 


Controllable pacsr 


1962 


Synchronous pacer 


1963 


Demand pacer 


1965 



HISTORIOGRAPH - CARDIAC PACEMAKER 



APPENDIX K. EMI SCANNER 



INTRODUCTION 



The EMI scanner, invented by G. N, Hounsfield of EMI Ltd. , is a computerized 
transverse axial tomographic (CTAT) device. (Tomography is derived from the Greek 
word meaning "to write a cut". ) Tomographic devices produce three-dimensional images 
by examining separate two-dimensional slices of an object. In CTAT, a series of trans- 
mission measurements are made with a narrow beam of X-rays traversing the selected 
section parallel to the faces. By taking a number of projections considerably greater 
than the number of resolution felements within the slice, an overdetermined set of equa- 
tions can be obtained which are solved to produce a reconstructed picture. Within allow- 
able radiation exposure limits, the amount of information which can be obtained with 
computerized axial tomography is much greater than that obtainable with conventional 
X-ray images. Since unwanted images below or above the plane of interest are eliminated, 
the CTAT scans can be interpreted much more easily than the conventional radiograph. 
In applications such as brain radiography where the skull shields the volume of interest, 
CTAT comprises a unique capability for noninvasive radiography. 

The clinical value of the EMI brain scanner was demonstrated in a group of case 
studies carried out by Dr. Ambrose at the Atkinson Morley Hospital in England with a 
prototype before the first commercial unit was installed at Mayo in June, 1973. Shortly 
after the EMI scanner became commercially available. Dr. Ledley at the Georgetown 
University Medical Center expanded the capability of the technique with the ACTA body 
scanner. However, the clinical value of body CTAT scanners has not been established 
yet. Clinical evaluations are in progress at several locations, including the Mayo Clinic, 
Georgeto'wn University Medical Center, the Mallinckrodt Institute of Radiology, and 
Northwich Park Hospital in England. Evaluations presented at the Radiology Society of 
North America Conference, December 1-4, 1975, show that the faster body scanners now 
available will almost certainly improve the situation by minimizing loss of resolution due 
to body motion. 

By the late 1960's, it seems that the stage was set for the development of CTAT. 
Within a period of less than a decade, several investigators in widely different fields 
including radiology, astronomy, and electron microscopy arrived at variations of the 
CTAT concept more or less independently. The principal element in the timing appears 
to be that computer development and digital data-handling and display techniques had 
advanced to the point that such development was possible. It is difficult to see how addi- 
tional medical research support could have advanced the innovation as much as 5 years. 

The Positron scanner has not been included in this history. Although it is also a 
three-dimensional radiographic technique ■which in many instances complements or serves 
a function similar to the EMI scanner, it does not have a noninvasive capability and has a 
developmental history sufficiently divergent from the EMI scanner as to be outside the 
present scope. Three-dimensional imaging by Fresnel aperture coding is also an 
interesting technique for imaging radioisotope distributions, but this too is outside the 
scope of this study. 



K-2 

The innovation of Hoansfield represents a confluence of scientific and technological 
development in four fields: computers, mathematics, radiology, and instrumentation. 
These channels, the significant events within each, and their interactions comprise this 
study. In accordance with the ground rules, events before 1945 are included only if they 
are considered decisive or if a brief early history is required to give continuity to events 
after 1945. 



HISTORICAL ACCOUNT 



Computers 



The automatic digital computer vi^as first conceived by Charles Babage in the mid- 
1800's. However, the manufacturing technology of the time was not capable of producing 
the precision parts required for his mechanical calculating machine. The first usable 
machine was not built until the early 1900' s ■when Herman Hollerith developed a punched 
card computer to mechanize the U. S. census counts. As with electronics in general. 
World War II provided a major boost to computer development for military applications. 
The first electronic on-line digital computer was Eniac, built by the University of 
Pennsylvania in 1946 and installed at the Aberdeen Proving Ground. This machine cal- 
culated the trajectory of a projectile while it was still in flight (Bylinsky, 1975). 

The basic functions and hardware of computers have changed little since the late 
1940' s, but the reductions in cost per calculation and in the difficulty in programming 
have been enormous. In the two decades starting at about 1950, the number of scientific 
computations which could be performed per dollar had increased by about a factor of 
100,000 (Streeter, 1974), which represents a near doubling every year in inflation- 
adjusted dollars. In addition, major reductions in application programming effort 
resulted from improved language and program libraries. No attempt will be made to 
list all significant computer developments since 1945. The historiograph will instead be 
limited to those developments that can be directly related to CTAT. The pioneering 
research at Bell Telephone Laboratories of Bardeen and Shockley on surface phenomena 
and Bardeen and Brattain (1948) on the invention of the triode transistor created a new 
era of electronics. Another major event leading to inexpensive minicomputers ■was the 
development of the integrated circuit about 1958 by Kilby at Texas Instruments and Noye 
at Fairchild (Klieman, 1966). The invention of the microproce sser chip by Hoff at Intel 
in 1969 (Bylinsky, 1975) is important to the high-information- rate systems under current 
development. 

During the 1950' s, the on-line computer installations were not common in the 
laboratory. However, the development of real-time computer systems for airline 
reservations, military command and control, and industrial processes, as well as the 
increasing programming capability of scientists, led to the requirement for automated 
experimental data handling by the late 1960's. The rapid expansion in availability of the 
on-line ininicomputers with complementary data acquisition and display hardware played 
an important role in the economic viability of computerized axial tomographic devices. 

Much of the pioneering development on digital image processing was done under the 
auspices of NASA to meet the image-processing requirements of pictures returned from 



K-3 

space vehicles (Nathan, 1966). Some of these concepts which have also proven to be 
useful in medical imaging application are: contrast enhancement, noise suppression by- 
attenuation of the high-frequency components in the Fourier-transformed images, 
partial elimination of low-frequency components to enhance fine details and edges, and 
automated pattern recognition (Lipkin, 1970). The significance of digital pattern recog- 
nition to CTAT is emphasized by the fact that both Hounsfield and Ledley indicate that 
their background in this area played an important role in their CTAT innovations. 

In the new developments toward real-time tomography, high-speed digital computer 
technology becomes even more significant (Robb, 1973). Analog-to-digital converters 
capable of 10 to 100 MHz have been developed at TRW (James and Ogawa, 1973, 1974), 
With these instruments, data-processing rates consistent with real-time CTAT are 
practical. 



Mathematics 



The mathematical basis for digital image reconstruction techniques started with 
the Austrian mathematician J. Radon. In 1917 he published a paper proving that a two- 
or three-dimensional object can be uniquely reconstructed from the infinite set of all its 
projections. However, substantial development of mathematical models capable of pro- 
viding accurate three-dimensional reconstruction had to wait for the modern digital 
computer. 

Over a dozen computer algorithms for image reconstruction have been proposed 
in the past 5 to 10 years. These fall into three main classes: the convolution methods, 
the Fourier methods, and the series expansion methods. A fourth class, the summation 
method, which includes classical tomography, might be included, but it does not share 
with the others the property of producing an exact mathematical solution with perfect 
experimental data. The Algebraic Reconstruction Technique (ART), published in 1970 
by Gordon, Bender, and Herman as a three-dimensional electron-microscope imaging 
technique is an example of the series expansion method. The original EMI scanner used 
this general method. From 1970 to the present, several variations, refinements, and 
noise- suppre ssion modifications have been developed by Johnson (1973), Herman (1973), 
Gilbert (1972a, b), and Goitein (1971). The theory of the convolution method was first 
developed by Bracewell and Riddle (1967) and later implemented by Ramachandran and 

Lakshminarayan (1971). The Bracewell and Riddle research was directed to three- 
dimensional astronomical reconstruction and the Ramachandran model was aimed at 
electron microscopy. Cho(1974), Budinger ( 1974), Vainshtein ( 1 973), Smith (1973), 
Gilbert (1972), Chesler (1972), Shepp (1973), and Lee (1973) have contributed variations 
and improvements to the basic method. The Fourier method was developed by DeRosier 
and Klug (1968). Additions to this method have been made by Crowther (1970), Budinger 
(1971), Lake (1971), Peters (1973), and Keyes (1973). 

In general, the convolution methods are most economical in computing time and 
require fewer views. However, if the projected images of an object exhibit high contrast 
or are contaminated with noise, the algebraic reconstruction algorithms usually produce 
the best reconstructions. All of the classes appear capable of reconstructing images 
with a resolution consistent with the physical limitations of the input and can be capable 
of enhancing large- or small-scale patterns in the reconstruction. However, pattern 
enhancement and psychology of pattern recognition have not been emphasized in CTAT 
as yet. 



K-4 

Instrumentation 



The scintillation counter is the preferred detector with most tomographic scanners. 
While the scintillation screen was available even at the time of Roentgen's initial work, 
it was not until the development of efficient photomultiplier tubes (Engstrom, 1947) that 
scintillation counting was advanced from the laborious visual counting of light pulses on 
a screen to the automated counter. A major advance in scintillation counters was made 
by Kallmann (1947), who found that large transparent blocks of naphthalene were effec- 
tive in producing scintillations. Bell (1948) showed that anthracine was about five times 
as effective as naphthalene, and Hofstradter (1948) discovered that the sodium iodide 
crystal activated with thalium was even more efficient, and, in addition, had a much 
larger photoelectric absorption cross section. 

Because the precision capability of CTAT is inherently high and fast scan speeds 
are needed, the counting statistics desired are correspondingly stringent. The 250- 
nanosecond decay time of Nal(Tl) is a disadvantage if the 10-MHz-or-faster digitizers 
currently available are to be fully utilized. The Sn-loaded plastic scintillators (NE 140), 
developed by Nuclear Enterprises, combine high photoelectric absorption with a 3- 
nanosecond decay time. Semiconductor detectors, such as Ge, Si, CdTe, Hgl2, and 
GaAs, are also available. Large charge -coupled-device (CCD) arrays and xenon multi- 
grid arrays may play an important role in fast, real-time CTAT. 

A further improvement in resolution could be realized with a suitable mono- 
energetic or narrower energy-band X-ray source. The laser-plasma X-ray source cur- 
rently in the research stage has promise, but these X-ray emissions are still too soft to 
be generally useful. 



Radiology 



The history of radiology begins with the discovery by Roentgen in 1895 of the 
emission of a penetrating radiation from a highly evacuated discharge tube. With excep- 
tional insight. Roentgen not only observed the radiation, and experimentally determined 
many of the macroscopic characteristics of X-ray interactions, but he foresaw many of 
the medical and physical applications. Within the next two decades radiography became 
established medical technology, and in the early 1920' s consideration was already being 
given to methods for circumventing the basic two-dimensional limitation of the radio- 
graph. A tomographic technique for radiography was first described by Bocage in 
France in 1921, but a practical model for medical use was not developed until an Italian 
(Vallebona, 1933) succeeded in imaging objects in a selected plane while blurring objects 
outside this plane into the background. The X-ray source was moved in one direction 
while the photographic film was simultaneously moved in the opposite direction, with the 
patient in between. The fulcrum of the motion described by the source and film was 
maintained in the plane of interest and stayed effectively "in focus". Various modifica- 
tions and improvements of this basic technique constitute the field of conventional tomog- 
raphy. Oldendorf replaced the X-ray film with a nuclear detector and reconstructed 
simple images in 1961. The classical or summation tomography method received addi- 
tional impetus from the development of a gamma-ray camera Tvith simultaneous readout 
of several planes (Anger, 1966). A computerized version of classical tomography with 
digital processing and displays was introduced in a scanner at the University of 
Pennsylvania medical school (Kuhl, 1968). 



K-5 

The development most directly related to present CTAT for medicine was reported 
in 1963 by Cormack, a physicist. His experiments with a (CTAT) scanner using a 
Geiger tube detector and a Fourier unfolding scheme represent the first workable 
instrument. Unfortunately, this work was reported in the Journal of Applied Physics 
and appears to have received little attention from the medical community. In a Yale 
University symposium on tomographic imaging held in 19V2 (Freedman), no reference 
was made to Cormack' s research. In fact the only reference to CTAT was an "in 
absentia" correspondence from Hounsfield. 

G. N. Hounsfield of the Central Research Laboratory of EMI started development 
of the EMI scanner in 1967, and produced his first crude pictures, using a gamima-ray 
source, at the end of 1967. Hounsfield's initial insights to the potential of the CTAT 
scanner concept started with earlier pattern-recognition studies at EMI. These included 
investigation of handwriting comparison by digital processing of the patterns. He was 
aware of the classical tomographic methods and felt that much better information could 
be obtained through computerized techniques. The work by Cormack was not known to 
the EMI group until later in the development. 

The British Department of Health appointed four medical advisors to evaluate the 
EMI scanner development on a continuing basis. Dr. J. Ambrose served in this capac- 
ity, advising on the clinical value that CTAT might have on brain-tumor diagnosis. The 
British Department of Health paid for the construction of the first prototype installed for 
Ambrose at the Atkinson Morley Hospital in 1971. By 1972, when information on the 
EMI scanner was first published, Ambrose had established its diagnostic importance 
with about 150 clinical cases. Another advisor. Dr. J. W. Bull, at the Institute of 
Neurology, National Hospital for Nervous Disorders, Queen Square, London, received 
the second prototype, and the first commercial instrument was delivered to Mayo in 
June 1973. The degree of medical acceptance of the EMI brain scanner is seen from the 
fact that in a time period of 3 years since the first commercial unit, about 500 of this 
type of instrument have now been sold. 

At the inception of the EMI scanner development, no on-line minicomputer capable 
of handling the data processing was available, and the intention was to use telephone - 
line links from remote stations to central computers. The rapid development of mini- 
computers in the late 1960's and early 1970' s allowed the change to the much more 
convenient and economical present systems. It is doubtful if the original concept would 
have attained the wide acceptance of the present self-contained instrument. 

Shortly after the EMI scanner reached the market, Dr. R. S. Ledley of the National 
Biomedical Research Foundation, Georgeto'wn University Medical Center, developed the 
ACTA body scanner. As with Hounsfield, Ledley' s immediate previous background was 
with digital pattern recognition. Ledley's instrument included stroboscopic capability 
to permit CTAT scans on organs having periodic motion. 

The clinical acceptance of the body scanner cannot yet be considered as an estab- 
lished fact. Unlike the brain scans, where the surrounding skull makes c(6nventional, 
noninvasive radiographs almost useless, the body scanner must be compared with other 
acceptable techniques. In addition, variations of the norm are much greater in other 
organs than in the brain. The first report of 119 body-scanner cases from Georgetown 
University did not pinpoint a unique usefulness of the device. Clinical trials with the 
EMI body scanner are beginning in the U. S. at the Mayo Clinic and the Mallinckrodt 
Institute of Radiology, and in England at the Northwich Park Hospital {Baker, 1975). 



K-6 

The main new developments in CTAT are in the direction of faster devices for 
real-time tomography of />? 1//V0 organs. According to Dr. E. H. Wood (1975) of the 
Mayo Clinic, 0, 01-second exposure times are needed for cardiovascular dynamics 
studies. Mayo plans to build a tomographic movie camera capable of 60 such exposures 
per second. 



REFERENCES 

Ambrose, J. A.E., and Hounsfield, G. N. , North Am. Radiological Soc. Meeting, 
Chicago (1972). 

Anger, H. O., in Instrumentation in Nuclear Medicine (Hine, G. J., and Sorenson, J. A., 
eds.). Academic Press, New York ( 1 974), Vol 2, 61-100. 

Baker, H. L. , Jr., Campbell, J. K., Houser, O. W. , et al. , Mayo Clin. Proc. , 49^, 
17-27 (1974). 

Baker, H. L. , Jr., Chief Radiologist of Mayo, Interview ( 1975). 

Baker, H. L. , Jr., Campbell, J. K. , Houser, O. W. , et al. , Mayo Clin. Proc, 49, 
17-27 (1974). 

Bardeen, J., and Brattain, W, H. , Phys. Rev., 73, 230-231 (1948), 

Bell, P. R. , Phys. Rev., 73, 1405 (1948). 

Bracewell, R. N. , and Riddle, A. C. , Astrophys. J,, 150 , 427-434(196 7). 

Budinger, T. F. , Ph.D. Thesis, University of California, Berkeley (1971). 

Budinger, T. F. , and GuUberg, G. T. , Lawrence Berkeley Laboratory Report LBL- 
2146 (1974). 

Bylinsky, G. , Fortune (November, 1975). 

Chesler, D. A. , in Tomographic Imaging in Nuclear Medicine (Freedman, G. S. , ed. ), 
Soc. Nucl. Med. , New York, 176 (1972). 

Cho, Z. H. , Ahn, I., Bohn, C. , and Huth, G. , Phys. Med, Biol. (1974), in press. 

Cormack, A. M., J. Appl. Phys., 34, 2722-2727 (1963). 

Crowther, R. A., DeRosier, and Klug, A., Proc. Roy. Soc. (London), A317 , 319-340 
(1970), 

DeRosier, D. J., and Klug, A., Nature, 217 , 130-134(1968). 

Electronics, 48, 34-38 (July 10, 1975), 

Engstrom, R. W. , J. Opt. Soc. Amer. , 37, 420 (1947). 



K-7 

Freedman, G. S. (ed. ), Proceedings of a Symposium on Tomographic Imaging in 
Nuclear Medicine , Soc. Nucl. Med., New York (1972). 

Gilbert, P.F.C., J. Theoret. Biol., 36_, 105- 1 17 ( 1972a). 

Gilbert, P. F. C., Proc. Roy. Soc. (London), Ser B, 182, 89-102 (1972b). 

Goitein, M. , Nucl. Instr. Methods, 101(3), 509-518(1971). 

Gordon, R. , Bender, R. , and Herman, G. T. , J. Theoret. Biol., 29, 471-481 (1970). 

Herman, G. T. , Lent, A., and Rowland, S. , J. Theoret. Biol., 42(1), 1-32 (1973). 

Hofstadter, R. , Phys. Rev., 74, 100 (1948). 

Hounsfield, G. N. , Patent Specification 1283915 (EMI Scanner), the Patent Office, 
London, England (1972). 

Hounsfield, G. N. , Interview. 

James, C. E., and Ogawa, K. K. , National Telecommunications Conference (Fall 1973), 

James, C. E., and Ogawa, K. K., National Telecommunications Conference (Fall 1974). 

Johnson, S. A., Herman, G. T. , Robb, R. A., Greenleaf, J. F. , Ritman, E. L. , Lee, 
S. L. , and Wood, E. H. , Mayo Clinic ms report (1973). 

Kallmann, H. , Natur und Techaik ( July, 1947). 

Keyes, J. W. , Jr., and Simon, W. , USAEC Report CONF-730627, 190-201(1973). 

Kilby, J. S. , U. S. Patent 3, 1 38, 743 (June, 1964). 

Kleiman, H. S. , Dissertation, George Washington University (1966). 

Knight, K. E. , Datamation (September, 1966). 

Kuhl, D. E., and Edwards, R. Q. , Radiology, 9J_(5), 975-983 (1968). 

Lake, J. A. , in Proc. Electron Microsc. Soc. Amer. 29th Annual Meet. (Arceneaux, 
C. J., ed. ), Claitor's Pub. Civ., Baton Rouge, 90-91 (1971). 

Ledley, R. S. , et al. , Science, 186 , 207 (July 12, 1974). 

Lee, D. E. , Keyes, J. W. , and Simon, W. , in Application of Optical Instrumentation in 
Medicine II, Proc. Soc. Photo. Optical Instr. Eng. (1973), in press. 

Lipkin, B. S. , and Rosenfeld, A., Picture Processing and Psychopictorics, Academic 
Press, New York (1970). 

Nathan, R. , J. P. L. Tech. Rep. No. 32-877 (January 5, 1966). 

Oldendorf, W. H. , IRE Trans, on Bio Med Electronics, BME-8, 1, 68-72 (January, 1961) 



K-8 

Peters, T. M. , Ph.D. Thesis, University of Canterbury, New Zealand ( 1973). 

Peters, T, M. , Smith, P. R. , and Gibson, R. D. , Brit. J. Radiol., 46, 314-317(1973). 

Radon, J., Bar. Saechs. Akad. Wiss. Leipzig, Math. Phys. Kl, 69, 262-277 (1917). 

Ramachandran, G. N., and Lakshminarayan, A. V. , Proc. Natl. Acad. Sci. U. S. , 
68(9), 2236-2240 (1971). 

Robb, R. A., Johnson, S. A., Greenleaf, J. F. , Wondrow, M. A., and Wood, E. H. , 
Proc. of the Soc. of Photo-Optical Instrumentation Engineers, 40, 11-26 (August, 1973). 

Roentgen, W. C. , Erste. Mitt. Sitzssber. Phys. -Med. Ges. Wurzburg, 137 (December, 
1895). 

Schmidlin, P. , in Medical Radioisotope Scintigraphy, 1972, International Atomic Energy 
Agency, Vienna, 409-417 (1973). 

Schmidlin, P., Nucl. Med., n_, 1-16 (1972). 

Schockley, W. , Electrons and Holes in Semiconductors, Van Nostrand, Princeton, N. J. 
(1950). 

Shepp, L. A. , IEEE 1973 Nucl. Sci. Symp. , in press. 

Smith, P. R. , Peters, T. M. , Bates, R. H. T. , J. Phys., A6, 361-382 (1973). 

Streeter, D. N. , The Scientific Process and the Computer, Wiley, N. Y. (1974). 

Vainshtein, B. K. , Usp. Fiz. Nauk, 109 , 455-497(1973). 

Vallebona, A., Fortche, a.d. Geb. d. Roentgen-strahlen, 48, 599-605 (1933). 

Wood, E. H. , Chairman of the Mayo Biophysical Sciences Unit, Interview (1975). 



INST 



f 
I 



.1947 Erl 



1947 K 



1948 E 



19' 



INSTRUMENTATION 



MATHEMATICS 
IMAGE RECONSTRUCTION 



RADIOLOGY (including SCANNER) 



COMPUTERS 



-1917 Radon Proof ol image reconstruction 
(rom an infinite set of projec- 



1947 Engstrom Development of 
photomultiplier 

DV/S/U/P/C 



1947 Kallmann Scintillations from napfitha- 
lene crystals 

NR/S/W/P/M 



1948 Sell Scintillation counter based on anif>ra- 
cene crystals 

MR/S/U/G/GL 



■ 1948 Hotsiadter Discovery of Nal(Tl) scint.l 
MR/S/U/P/M 






CODES 


NR 


Nonmission-Oriented Research 


MR 


Mission-Oriented Research 


DV 


Development 


S 


Signilicant Event 


D 


Decisive Event 


U 


U.S. Event 


W 


Foreign Event 


G 


Government Funding 


P 


Private Funding 


M 


Medical School - Hospital - 




University 


1 


Research Institution 


GL 


Government Laboratory 


C 


Company 



-1970 Gordon Development of algebraic recon- 
struction technique 

MR/S/U/P/M 



— 1971 Ramachandran Numerical implemeni 
tion of convolution 
method 
MR/S/U/P,G/M 



1895 Roentgen Discovery and characteriza- 
NR/D/W/P/M 



- 1933 Vallebon; 



t tomographic sysK 
DV/S/W/P/M 



1961 Oldendorf Patent on tomographic 
vice with nuclear deiec 
and rotating obiect 
DV/S/U/P/M 



1 

1953 Cormack Developed first complete 
workable CTAT technique 
MR/S/U/P,G/M 
(panial AEC support) 

- 1966 Anger Developed multi-view gamma 
DV/S/U/G/GL 



1967 Hounsfield First crude El^l scanm 
(with gamma source! 
MR/D/W/P/C 



f1947 Eckert Enia, first electronic "on-l 
digital computer 

DV/S/U/G/M 



1968 Kuhl Developed classical tomographic 
scanner with digital processing 
for clinical use 

DV/S/U/G/M 



1971 Hounsfield Developed first clinical 
prototype 

DV/D/W/G,P/C 



1972 Ambrose Publication of first clinica 
evaluation of EMI brain 



DV/S/W/G,P/M 



-1974 Ledley Development of ACTA body 
DV/S/U/P/M 



1948 Bardeen I 



1958 Noyce li 
Kilby 



DV/D/U/P/C 



m of integrated ci 
DV/S/U/P/C 



■1960-NASA Development of digital image 
1970 processing tediniques 

DV/S/U/G/G 



I Hoff Development of the microprocessor 
DV/S/U/P/C 



HISTORIOGRAPH-EMI SCANNER 



L-1 
APPENDIX L. POLIO VACCINE 

INTRODUCTION AND SUMMARY 



Poliomyelitis was not recognized by the medical profession as a distinct clinical 
entity until the end of the 18th and the early 19th centuries. At first only sporadic cases 
were reported, but by the first half of the 19th century, small outbreaks had been de- 
scribed. The disease assumed importance late in the 19th century when suddenly it 
changed from bring sporadic and endemic, and became an epidemic scourge, particu- 
larly in Scandinavia and the United States. The discovery of the etiologic agent in 1908 
by Landsteiner and Popper was folio-wed by a burst of research activity on the part of in- 
vestigators in S-weden, Germany, France, and the United States. Early optimism con- 
cerning the feasibility of producing an effective vaccine faded as the problems and diffi- 
culties in working with the agent in the only available experimental animal, the monkey, 
were encountered. Laboratory investigations for almost a generation were focused on 
a study of the disease induced by intranasal installation of the virus in monkeys, using 
a single strain -which had become highly neurotropic as a result of many serial passages 
in monkey central nervous system (CNS). These studies led to the incorrect belief that 
in humans poliovirus entered the nose and travelled directly to the CNS via the olfactory 
path-way. 

Reorientation of thinking about the pathogenesis of the natural infection began in 
the 1930' s when gradually it was shown that the intestinal tract is the primary site of 
virus multiplication, and that most infections are inapparent, localized to this area, and 
result in long-lasting immunity. Serologic surveys during the late 1940' s and the 1950' s 
in areas -where the sanitary environment was poor and the paralytic disease rare, then 
showed that in such situations the virus circulated widely, children were infected and 
immunized in the first few years of life, and no epidemics occurred. In economically 
advanced countries, in contrast, improvements in sanitation prevented early exposure 
and infection, allo-wing children to remain unprotected so that -when a virulent strain -was 
introduced it could spread rapidly among the pools of susceptibles that had been built up. 
This resulted in epidemics of increasing size and severity. 

The development of effective vaccines -was made possible by the better understand- 
ing achieved about the nature of the infection, its pathogenesis and immune mechanisms, 
and the role of serum antibodies in blocking the viremic phase which -was found to pre- 
cede CNS invasion. The demonstration that the virus exists in three antigenically dis- 
tinct serotypes -was essential. The most important technical breakthrough -was the dis- 
covery by Enders, Weller, and Robbins that poliovirus could be propagated in vitro in 
tissue cultures of monkey cells derived from nonnervous tissue. 

In the early 1950's, both formalin-inactivated and live attenuated virus vaccines 
were developed. The killed vaccine of Salk was the first to be licensed, in 1955. It was 
shown to induce serum antibodies which protected against the paralytic disease, and be- 
tween 1955 and 1960 its use resulted in a remarkable decline in poliomyelitis in the 
U. S. Kopro-wski was the first to test a live attenuated virus vaccine -when in 1952 he 
immunized ZO volunteers -with a rodent-adapted strain of Type II administered orally. 
At the same time, Sabin as well as Kopro-wski and Cox began their efforts to develop a 
live attenuated virus vaccine by serial passage in tissue cultures of monkey cells. 
AviruLent strains of all three types were successfully prepared and found to be immuno- 
genic by the oral route. After exhaustive tests in animals, folio-wed by trials of in- 
creasing size in humans, the Sabin strains were accepted as the optimum ones for 



L-2 

human use. They were licensed in 1960-1961. Because the oral vaccine simulates the 
natural infection, has greater immunogenicity, induces antibodies more rapidly, and has 
the advantage of ease of administration, it has gradually supplanted the Salk vaccine in 
the U.S. 

The fact that a popular president, Franklin D. Roosevelt, -was himself a polio vic- 
tim, occasioned public interest and support of poliomyelitis research in the U. S. The 
main agency for support was the National Foundation for Infantile Paralysis (NFIP). 
The NFIP had grown out of a small fund-raising group which had been organized as the 
President's Birthday Ball Commission in 1934, the year after Roosevelt had taken office 
as president. The Commission v/as a successful enterprise, but in 1938 was phased out 
and replaced by the NFIP, headed by Basil O'Connor, a former law partner of Roosevelt 
before the future president had suffered a devastating attack of paralytic poliomyelitis. 
O'Connor was a man of enormous energy, drive, and purpose. He succeeded to an un- 
precedented degree in raising huge sums of money from a willing public to support re- 
search on poliomyelitis and to provide care for all the unfortunate persons who devel- 
oped the disease. Gradually the Foundation, through its committees and grants, 
assumed a commanding position in guiding the direction of research and particularly 
the developnnent of poliovirus vaccines. However, the triumph of successful control of 
poliomyelitis came about primarily as a result of the imagination and genius of dedi- 
cated scientific investigators such as Paul, Enders, Salk, Sabin, and many others. The 
contribution of the NFIP, which generously supplied funds for the enterprise, was that 
it made it possible to reach the final goal sooner than would otherwise have been the 
case. 



HISTORICAL ACCOUNT* 



Etiology 



The first recognizable description of paralytic poliomyelitis appeared in 1789 in 
a textbook on diseases of children by Michael Underwood, an English physician. How- 
ever, recorded examples of lameness and v/ithered limbs dating from ancient times in- 
dicate that the disease has existed in endemic form since the earliest recorded history, 
and probably since earliest evolutionary times. For the first time small outbreaks 
were reported in England and in the United States in the early 19th century (Paul, 1971). 
All of them mainly involved children under 2 to 3 years of age. The cause was obscure, 
and variously ascribed to "teething" or "debility (in) children previously reduced by 
fever". 

By I860, Jacob Heine, a German orthopedist, was able to produce a comprehen- 
sive monograph on "infantile paralysis". His interest was primarily in the late de- 
formities which followed a paralytic attack, but he included an accurate clinical de- 
scription of the acute disease. Heine postulated that the anterior segment of the gray 
matter of the spinal cord was the site of the pathologic process. This interpretation 
was not generally accepted and there followed a period of almost 30 years of contro- 
versy over whether the central nervous system (CNS) was the primary site of injury or 
was only secondarily affected. Finally, in 1870, the French investigators Charcot and 



*The reader is referred to A History of Poliomyelitis by J. R. Paul, Yale University Press (1971) for a complete account of polio- 
myelitis and its control. 



L-3 

Joffroy, using new histological techniques, confirmed Heine's theory and demonstrated 
that in fatal cases the characteristic lesions of paralytic poliomyelitis were in the neu- 
rons of the anterior horns of the gray matter of the spinal cord. Still the question per- 
sisted as to whether the neurons were the primary site of attack or were damaged as a 
results of inflammatory lesions in the surrounding tissue. This issue was finally re- 
solved by the studies of Rissler, who in 1888 described the specific neuronal changes 
and pointed out that they could occur independently of interstitial changes. 

In the late 19th century, a marked change occurred in the behavior of paralytic 
poliomyelitis. Whereas it had remained a sporadic disease of young children with oc- 
casional small clusters of cases, it suddenly began to appear in epidemics of increasing 
size and severity. This happened first in the Scandinavian countries, particularly in 
S-weden. The eminent S-wedish professor of pediatrics, Karl Oscar Medin, investigated 
an epidemic of 44 cases in Stockholm in 1887, and confirmed the emerging view that 
poliomyelitis was caused by an infectious agent. It was Medin who brought poliomyelitis 
to the attention of the medical world when he discussed his findings at the Tenth Inter- 
national Medical Congress in Berlin in 1890. He was a great clinician and an impressive 
person; the story he told of the sudden appearance of epidemics of infantile paralysis, 
his astute observations on the clinical features and on the nature of the disease, made a 
strong impression on the European medical community. For some years the disease 
was known as Heine-Medin disease in recognition of the major contributions of these two 
men. An important step in understanding the epidemiology of the disease and the way in 
which it spread came from the studies of Medin' s pupil, Ivar Wickman { 1907), who was 
the first to recognize that not all infections resulted in paralysis. In a meticulous in- 
vestigation of an outbreak in a small town in Sweden, he traced the chain of infection of 
each case and demonstrated that nonparalytic and abortive forms of the infection ■were 
the major source of dissemination of the agent. 

The latter part of the 19th century was a period of sudden flowering of knowledge 
in the sciences of pathology and bacteriology. The discoveries of Virchow, Koch, 
Pasteur, and others opened a vi^hole new world in the field of infectious diseases. A 
inajor landmark was the demonstration by Pasteur in 1881 that the etiologic agent of 
rabies, which could not be cultivated on artificial media that supported the growth of 
bacteria, could nevertheless be recovered by intracerebral inoculation of rabbits with 
CNS material from infected cases. Pasteur called the agent a virus. It was the first 
time that the term had been used. 

During this period it v/as common practice for bacteriologists to use porcelain 
filters of various grades to clear their cultures of contaminating particulate matter. 
Following passage of the medium through the filter, it was considered to be free of or- 
ganisms. In 1892, the Russian botanist Ivanowski found that the filtrate of fluid pre- 
pared from infected tobacco leaves contained the infectious agent of mosaic disease of 
tobacco (Clifton, 1950). In 1898, Loeffler showed that the agent responsible for foot and 
mouth disease in animals also passed through a pocelain filter (Clifton, 1950). At this 
point the phrase "filter passing virus" ^vas coined to describe those infectious agents 
■which ■were so small that they could not be resolved by light microscopy. 



Discovery of Poliovirus 

At the turn of the century, the techniques for studying viruses were still crude and 
limited largely to inoculation of experimental animals. Nevertheless, by the first decade 
of the 20th century the viruses responsible for rabies, smallpox, vaccinia, and foot and 



L-4 

mouth disease had been identified. In 1908, working in the Allgemeine Krankenhaus in 
Vienna, Landsteiner and Popper attempted to recover the etiologic agent of poliomyelitis. 
Using a bacteria-free homogenized suspension of spinal cord obtained from a fatal case, 
they injected rabbits, guinea pigs, and mice, all of which remained healthy. They also 
injected two monkeys intraperitoneally with the same specimen. One, a Cynocephalus 
hamadryas, succumbed 8 days later without apparent evidence of paralysis. The other, a 
Macaca Mulatta (rhesus), developed typical flaccid paralysis in both legs after an incuba- 
tion period of 17 days. The disease picture and course in this animal closely resembled 
paralytic poliomyelitis in man. Moreover, histologic examination of the spinal cords of 
both monkeys revealed extensive lesions which v^ere identical to those observed in hu- 
man poliomyelitis. 

The period 1909 to 1912 saw great activity and many discoveries in the several 
laboratories in Europe and in the U. S. which had taken up research on poliomyelitis. 
News of Landsteiner and Popper's discovery crossed the Atlantic quickly, and Simon 
Flexner, director of the Rockefeller Institute for Medical Research in New York City, 
immediately went to work to confirm the new findings. He and his colleagues recovered 
the virus from the spinal cords of two cases of fatal poliomyelitis and succeeded in 
passing the agent from monkey to monkey (Flexner and Clark, 1912; Flexner and Lewis, 
1910). In France, Levaditi began experimental work v/ith the virus in monkeys collab- 
orating at first with Landsteiner (1911). The Swedish investigators Kling, Wernstedt, 
and Pettersson at the State Bacteriological Institute in Stockholm took up where Medin 
and Wickman had left off, and -were the first clinical virologists to enter the field. They 
published a classic paper in 1912, in ■which they demonstrated for the first time the 
presence of the virus in nonnervous tissues. This was done by inoculating monkeys with 
throat and intestinal washings of living polio patients. They also reported the recovery 
of the virus from the oropharynx, trachea, intestines, and central nervous system in 
most of the 14 fatal cases examined. Unfortunately, the criteria used in reading the 
histological sections of monkey spinal cords was not very rigorous, -with the result that 
the Swedish findings were greeted with skepticism and actually dismissed by other in- 
vestigators including Flexner and Levi^is (1910), w^ho vi^ere greatly impressed by the 
neurotropism of the virus in the experimental host, the rhesus monkey. Nevertheless, 
in retrospect it is clear that Kling and his team did demonstrate poliovirus in nonneural 
sites in the human infection, even though all of the tests reported v/ere not acceptable. 
In addition, the results obtained by the Swedish group proved that Wickman' s theories 
concerning the clinical epidemiology of poliomyelitis were correct. The isolation of the 
virus from patients with mild inapparent infections demonstrated the importance of 
these individuals in the spread of the disease. 

As early as 1910, Flexner and Le-wis sho-wed that convalescent serum from mon- 
keys surviving the experinaental disease contained antibodies that would neutralize (in- 
activate) poliovirus; in the same year, Netter and Levaditi demonstrated that such neu- 
tralizing antibodies vi^ere also present in the blood of hunnans recovering from the 
disease. Wernstedt, a member of the Swedish team, was the first to postulate that, in 
general, immunity was acquired from inapparent infection, and that this accounted for 
the striking age incidence of poliomyelitis (Kling, Pettersson, and Wernstedt, 1912). 
Thus, more than 90 percent of overt cases of the disease occurred in susceptible infants 
and young children, older children and adults being resistant by virtue of previous in- 
apparent immunizing infections. 

Wernstedt' s theory was confirmed in the United States on the basis of field stud- 
ies conducted by Wade Frost ( 1913), an epidemiologist of the U. S. Public Health Ser- 
vice, during three successive summers in which polionayelitis was epidemic (Mason 



L,-5 

City, Iowa, 1910; Cincinnati, Ohio, 1911; Buffalo, New York, 1912). Frost collected 
blood Scimples from nine patients (Mason City epidemic) who had recovered from, attacks 
of what was assumed to be abortive poliomyelitis. Neutralization tests conducted by 
Anderson and Frost (1911) on these sera showed that antibodies to poliovirus were pres- 
ent in both the test sera and sera collected from "normal" adult controls. These find- 
ings suggested that a previous inapparent infection must have occurred to account for the 
presence of specific antibodies to the virus, Peabody and associates (1912), of the 
Rockefeller Institute, soon confirmed these findings and showed further that normal 
adults possess poliovirus antibody levels similar to those present in the sera of conva- 
lescent paralytic patients. 

Although there was evidence that infection -with poliovirus -was commonly inappar- 
ent, and that the virus could be spread by direct contact with persons having such infec- 
tions, as well as with paralytic cases, these facts were neither widely known nor gen- 
erally accepted by the scientific community. This was apparent in the drastic quarantine 
measures employed in New York City during the great 1916 poliomyelitis epidemic 
there, one of the largest ever recorded. 

During the period 1912 to 1917, when New York suffered several severe epidem- 
ics, clinical studies at the Rockefeller Institute Hospital were concentrated on the dis- 
ease as exemplified in hundreds of patients. Peabody, Draper, and Dochez published an 
exhaustive monograph on the subject in 1912. There were no attennpts to recover polio- 
virus from the patients. All experimental work at this time was carried out in Dr. 
Flexner's laboratory, where efforts were directed toward understanding the disease by 
studying it in monkeys, the only susceptible experimental host. Since monkeys -were 
not easily available, difficult to house and care for, and expensive, few laboratories 
in the United States were able to undertake research on poliomyelitis. The field was 
therefore dominated for the next t^vo decades mainly by Dr. Flexner and his group v/ho 
had at their disposal the modern well-equipped laboratories of the Rockefeller Institute, 
which had ample facilities for working with primates. Many facts about the experi- 
mental infection ■were uncovered, but, unfortunately, virtually all of the experiments 
were conducted with one strain of virus (MV). After reiany successive passages by the 
intracerebral route, this strain became exclusively neurotropic, and as was subse- 
quently learned, behaved very differently from naturally occurring poliovirus in the hu- 
man host. 

During this early period Dr. W. Lloyd Aycock of Harvard University was also a 
major figure in the field. He becaime an acknowledged authority on the epidemiology 
of poliomyelitis, but his name is associated primarily with "autarcesis", a term that 
he derived from two Greek words to describe non-specific resistance to the disease. 
This type of resistance, he believed, was the most important deterrent to the acquisi- 
tion of infection. He considered it to be the result of a process of immunological ma- 
turation which was distinct from specific immunity. His ideas were thus at odds with 
those of Wernstedt who, as early as 1912, recognized that inapparent infection is the 
mechanism by which immunity is acquired and that this accounts for the resistance en- 
joyed by the majority of the population. Ironically, it was Aycock and his colleague 
Kramer who provided the first laboratory evidence that Wernstedt' s viev/s were correct: 
in 1930 they published a paper in which they compared age specific immunity in urban 
and rural populations to poliomyelitis and to diphtheria, using a monkey neutralization 
test and the Schick test, respectively. The results paralleled one another to a remark- 
able degree, and the conclusion was drawn that immunity to poliomyelitis had been ac- 
quired by previous inapparent infection, and was not due to autarcesis. 



L-6 

Draper, in 1917, published a book on poliomyelitis in which he described a sys- 
temic phase of the human infection which precedes involvement of the CNS. Despite 
this, and despite the earlier work by Flexner and Lewis (1910), Kling et al. (1912), and 
others, in vi^hich virus had been recovered from nonneural sites, the concept of polio- 
virus as a strictly neurotropic agent gained increasing ascendency. In experiments in 
m.onkeys inoculated intranasally with the neuro-adapted MV strain, the virus was demon- 
strated to travel directly via the olfactory nerves to the central nervous system. These 
results were interpreted as indicating that the course of events in the natural infection 
in humans was similar, that the virus entered through the nose and spread only through 
nervous pathways to the brain. Flexner's concepts of the pathogenesis of the disease 
dominated the thinking of investigators working in the United States for almost a genera- 
tion. They even formed the basis of an ill-conceived approach to the prevention of the 
disease, following the demonstration by Armstrong and Harrison in 1935 that pretreat- 
ment of the olfactory mucosa of rhesus monkeys with sodiunn alum would prevent the 
paralysis that followed intranasal instillation of poliovirus. These results were con- 
firmed by other investigators using a variety of chemicals, and led to field trials in hu- 
mans using nasal chemical blockade as an approach to prophylaxis during epidemics of 
poliomyelitis. Several clinical trials (Armstrong, 1937; Tisdall, et al. , 1937) demon- 
strated that nasal blockade with chenaicals was ineffective in preventing naturally ac- 
quired poliomyelitis in humans; furthermore, it often had the unfortunate side effect of 
destroying the sense of smell. 



Studies in Man 



During the 1930's, at long last there was a return to a study of poliomyelitis in 
man. During an epidemic in Connecticut in 1931, Paul and Trask (1933) investigated 222 
families in which a paralytic case had occurred. They found a high incidence of minor 
illnesses in the younger family members, and estimated that abortive poliomyelitis oc- 
curred at least 6 times the rate of paralytic disease. They recovered poliovirus from 
the throat washings of two of the children with the minor illness syndrome. These were 
the first isolations of the virus from living patients in almost 20 years. Subsequently, 
in 1938, Trask, et al. , also recovered the virus from stool extracts of an infant with 
abortive poliomyelitis, 2, 14, and 25 days after onset. These findings together with the 
observation by Sabin in 1940 that the olfactory bulbs of patients dying of paralytic polio- 
myelitis did not reveal lesions comparable to those found in intranasally inoculated mon- 
keys, turned the tide in the thinking about the human disease. It became apparent that 
the infection is a systemic one involving primarily the intestinal tract, but also including 
the oropharynx. Invasion of the CNS is a relatively rare accident. This interpretation 
was strengthened by the report by Sabin and Ward in 1941, sho\ving that in fatal cases 
virus is found consistently in two sites — in the intestinal tract and in the CNS — but not 
in the olfactory apparatus. In the same year, Howe and Bodian (1941) demonstrated that 
stool filtrates from acute cases -were infectious for chimpanzees inoculated intranasally 
or orally. However, olfactory bulb lesions developed only in those inoculated intra- 
nasally; excretion patterns in the stools of chimpanzees were similar to those found in 
humans. Subsequently, cynomolgus monkeys (M. cynomolgus) were also found to be sus- 
ceptible to oral infection. These animals and chimpanzees proved useful in reexamin- 
ing the pathogenesis of poliovirus infection in experimental animals infected by a natural 
route — orally — rather than by an artificial method such as intranasal or intracerebral 
inoculation. 



L-7 

The return to investigations using freshly isolated, rather than laboratory adapted, 
strains of poliovirus also uncovered two vital points about this agent, namely, that it 
exists in more than one distinct antigenic type, and that there is no significant cross pro- 
tection between types. Burnett and Macnamara in 193 1 in Australia -were the first to 
publish a report indicating the existence of immunological differences between strains of 
poliovirus. This announcement -was greeted -with skepticism by some in the scientific 
community, but Paul and Trask (1933), using two strains recovered from patients during 
the 1931 epidemic in New Haven, Connecticut, repeated the Australian work and showed 
that there were at least two antigenically distinct types of the virus. Cross-immunity 
tests in monkeys performed by Bodian and others on the NFIP Typing Committee in 1949 
(Paul, 1971) confirmed these observations. At this time a collaborative program for 
the typing of 100 naturally occurring strains of poliovirus was organized by the NFIP. A 
nunaber of laboratories virere involved, and many hundreds of monkeys were used in 
cross-protection tests. The results indicated that polioviruses exist in only three differ- 
ent serotypes. This information vv^as vital to the development of effective vaccines 
against the disease, and to evaluation of immunity by serologic tests. 



Development of Polio Vaccine 



Efforts to develop vaccines against poliomyelitis actually began soon after dis- 
covery of the etiologic agent. Flexner and Lewis, in 1910, were the first to produce ac- 
tive immunity, using small doses of virus present in infected monkey spinal cords, and 
giving injections repeatedly over a period of weeks. The test monkeys thus immunized 
became resistant to intracerebral challenge with virulent virus. The method was risky, 
however, and paralytic disease sometimes occurred in the course of immunization. In- 
vestigators on both sides of the Atlantic therefore tried to prevent this complication by 
partially inactivating the virus with heat. In addition, formalin inactivation was tried by 
Romer in Germany as early as 1911, but this was unsuccessful, as was the case with a 
variety of other chemical inactivators which were examined. The problems encountered 
resulted in a dampening of enthusiasm for the -whole subject and efforts to develop vac- 
cines languished for the next two decades. 

In the mid-1930's, two groups of investigators undertook to prepare vaccines 
against poliomyelitis, using infected monkey spinal cord. In 1934, Brodie, working in 
Ne^v York, reported that formalin-inactivated virus was immunogenic in 20 monkeys and 
protected the animals against subsequent intracerebral challenge. On the basis of these 
findings, Brodie and Park in 1935 began a program in -which 3000 children were given 
the vaccine. In the same year, Kolmer, in Philadelphia, developed a live attenuated 
virus vaccine, using virus which had been passed serially in monkeys, theoretically to 
reduce its virulence, and by treating the infected-spinal-cord preparations -with sodium 
ricinoleate to further reduce — but not eliminate - all infectivity. Tests in 42 monkeys, 
2 adults, and 22 children were reported to prove the efficacy and safety of the prepara- 
tion and it was therefore distributed to the medical profession for clinical trials. When 
given to larger numbers of children, the results with both Brodie's and Kolmer's vac- 
cines proved disastrous, for a number of cases of paralytic poliomyelitis followed 6 to 
14 days after administration. Dr. James Leake, of the U. S. Public Health Service, 
described a series of such cases in 1935, and concluded that they render "undesirable 
the further use of poliomyelitis virus for human vaccination at this time". The blow 
was a devastating one to Brodie, who conann.itted suicide. It was also a blow to the 
cause of vaccine development, for the memory of the tragic events of 1935 made investi- 
gators shy away from the subject for almost 20 years. 



L-8 

The reasons for the failure of the Brodie and Kolmer vaccines are now clear: the 
preparations used were extremely crude, accurate quantitative evaluation of virus con- 
tent and inactivation were not possible with the methods available, nervous tissue is un- 
suitable as a substrate for a vaccine, and finally, it was not appreciated at the time that 
polioviruses exist in more than one serotype. 

A numiber of new discoveries in poliomyelitis research in the 1940's and 1950's 
contributed to set the stage for the development of the safe and effective vaccines that we 
now have. One was a better understanding of the nature of serologic immunity and its 
persistence. With Armstrong's discovery in 1939 that the Lansing strain of poliovirus 
(later found to be a Type II) could be adapted to rodents, a tool was available for sero- 
epidemiologic studies using the mouse neutralization test. These studies were instru- 
mental in explaining sonne of the puzzling features of the behavior of the disease, such 
as the absence of epidemics and the rarity of the paralytic disease in developing, largely 
tropical, countries. In investigating the problem, Paul and his colleagues showed that 
contrary to what had been assumed, the viruses are widely disseminated in such popula- 
tions which have poor sanitation and hygiene. Under these circumstances, infection and 
innmunity are acquired by virtually all children in the first few years of life, epidemics 
do not occur, and sporadic cases are limited to those under 5 years of age. With eco- 
nomic development and improved sanitation and hygiene, young children are protected 
from exposure and infection, and the number of susceptibles among older children and 
young adults builds up. An epidemic may then result when, during the summer, a viru- 
lent strain is introduced. Absence of immunity is also characteristic of populations 
living in isolated communities, as shown by Paul, et al. , in a classic investigation car- 
ried out among Eskimos living in Pt. Barrow, Alaska. The results indicated that a 
single exposure resulting in inapparent infection, induced neutralizing antibodies that 
persisted as long as 40 years. This suggested that lasting immunity could be induced 
by ingestion of live but avirulent, nonparalytogenic strains of the virus. 

Another significant observation which supported the feasibility of prophylactic im- 
munization was the consistent demonstration of a viremic stage early in the incubation 
period of the disease. Horstmann reported in 1952 on the isolation of poliovirus from 
the blood of orally infected monkeys and chimpanzees. Similar results vi^ere soon pub- 
lished by Bodian. In 1954, Horstmann, et al. , tested blood from family contacts of 
paralytic cases and reported that viremia -was regularly present during the incubation 
period or minor-illness form of the infection. Following reappraisal of the pathogene- 
sis of human poliomyelitis and recognition that a viremic phase occurs some days before 
the appearance of CNS signs, it became theoretically feasible to block access of the 
virus to the CNS by stimulating the production of circulating antibodies with a killed (or 
live attenuated) virus antigen. Such antibodies vi^ould neutralize the virus, prevent CNS 
invasion, and thus prevent the paralytic disease. 

All these observations were important in providing a rationale for the development 
of vaccines — both of the killed and live virus types. But the goal could not have been 
achieved without the technical bresikthrough that came with the discovery in 1949 by 
Enders, Weller, and Robbins that poliovirus could be grown in tissue culture. Until that 
time, the only source of the three types of poliovirus was monkey nervous tissue, an un- 
acceptable substrate because of its capacity to induce a demyelinating Type A encepha- 
litis. The observations of Enders, et al. , thus had a profound effect, not only in terms 
of the subsequent development of vaccines by Salk and by Sabin, but because they revo- 
lutionized the whole field of virology and led to the discovery of hundreds of new viral 
agents. Although living tissue fragments had been grown in vitro by Harrison as early 
as 1907, the Maitlands v/ere the first to propagate a virus in such cell cultures vi^hen in 
1928 they reported the growth of vaccinia virus in vitro (Paul, 1971). But tissue culture 



L-9 

did not become a practical method until antibiotics were available to control bacterial 
contamination. Not only did the new technique provide means for accurate quantitative 
assessment of virus growth and inactivation, but it also made possible evaluation of im- 
mune status and measurement of the effectiveness of vaccines by serologic tests. 

By 1950, v/ith improved understanding of the pathogenesis of poliovirus infections 
and the technical advances provided by the introduction of tissue-culture methods, the 
time ■was ripe to get on -with the task of preparing a vaccine. Accordingly, in 1951, a 
Committee on Immunization was appointed by the NFIP. It consisted of a number of 
leading research workers in the field*, who met and debated at great length over the 
next 2 years whether a killed or live attenuated virus vaccine was most suitable. Both 
Salk and Sabin v^ere members, and their vie'ws — and those of others on the committee — 
differed sharply. There was also concern about the ethics of testing vaccines in chil- 
dren, the only age group in which there were sufficient numbers of susceptibles to pro- 
vide a statistically adequate test. The NFIP vievired the caution of the scientists -with 
impatience, and to insure more rapid progress, a new committee, the Vaccine Advisory 
Committee, was appointed in 1953 (Paul, 1971). Its members, largely drawn from the 
public health profession, were specifically charged with the responsibility of pushing 
ahead with the inactivated vaccine on which Jonas Salk had been working for several 
years, and which the NFIP strongly favored. 

Salk -was in a particularly favorable position to undertake the mammoth task ahead. 
He had the advantage of extensive experience with the influenza -virus vaccine trials di- 
rected by Dr. Thomas Francis during World War II, under the aegis of the Armed 
Forces Epidemiological Board. Salk had also been a leader in the collaborative 
poliovirus-typing program of 1949 to 1950. 

By 1953 he was far enough along with work on an inactivated poliovirus vaccine 
that he and his colleagues at the University of Pittsburgh were able to publish prelimi- 
nary observations on active immunization of human subjects with tissue-culture grown 
virus inactivated by formalin. This was not a new method of vaccine preparation, since 
it had been tried on poliovirus as early as 1911 by Rbmer, and again in 1934 by the ill- 
fated Brodie. What was new vi'as the possibility of controlling the inactivation precisely, 
of measuring accurately the amounts of virus present, and of insuring the absence of 
residual live virus when the reaction was completed. As subsequent larger trials pro- 
ceeded in Salk's laboratory, with uniformly successful results in terms of safety and 
efficacy in inducing neutralizing antibodies in susceptible subjects, the NFIP moved 
ahead to organize, under the leadership of Dr. Thomas Francis, a massive, collabora- 
tive, controlled field trial in 1954. 

The highly successful results of the "Francis trial" were announced by the NFIP 
on April 12, 1955, amidst great publicity and fanfare (Paul, 1971). The vaccine was 
immediately licensed and released for use-by the medical profession. But tragedy 
struck again. Within 2 -weeks it became apparent that a few of the vaccine lots of one 
manufacturer had contained active virus; as a result, some 79 children developed para- 
lytic poliomyelitis and transmitted the disease to more than 100 contacts. The offend- 
ing lots of vaccine were quickly identified, and the procedures for production control 
were strengthened. After careful review of the situation, it was decided to go ahead, 
and no further such episodes occurred in the ensuing years, during -which millions of 



•Members of this committee included: Drs. David Bodlan, John F. Enders. Thomas Francis, Jr., Wm. McD. Hammon, 
Howard A. Howe, John R. Paul, Andrew J. Rhodes (of Canada), Joseph E. Smadel, Albert B. Sabin, Jonas E. Salk, 
Thomas B. Turner, and Antonio Ciocco. Representing the National Foundation were Mr. Basil O'Connor, 
Drs. Thomas M. Rivers, Hart Van Riper, Medical Director, and Harry Weaver, Director of Research. 



L-10 

doses were administered. In the period 1954 to 1959 when Salk vaccine was widely used, 
the annual attack rate of poliomyelitis in the U. S. fell from 13. 9 to 0. 6 per 100, 000 — 
a remarkable decline. 

The group of investigators who favored a live attenuated poliovirus vaccine from 
the beginning continued to work on this aspect throughout the 1950's. Koprowski (1952), 
taking a leaf from experimental work that Theiler had reported at an NFIP meeting on 
immunization in 1946, attenuated a strain of Type II poliovirus by serial passage in cot- 
ton rats. When given orally to 20 susceptible children, the vaccine induced an intestinal 
carrier state lasting several weeks, and high titers of neutralizing antibody appeared in 
the sera. This was the first trial of such a vaccine in humans. Koprowski pursued his 
lead further, but when practical tissue-culture techniques became available, serial 
passage of virus in primate nonneural cells quickly supplanted all other methods in the 
preparation of an attenuated vaccine. 

The major figure in the live-virus-vaccine field was Albert Sabin, who pushed 
ahead with enormous vigor. There was much to support the rationale of a live-virus 
vaccine which would simulate the immunity acquired naturally through inapparent infec- 
tion — i.e., the way in which most populations acquired their resistance to the disease; 
such vaccines could achieve this end without the risk of paralysis which infection with 
wild virus entailed. Studies on orally infected chimpanzees in 1946 by Melnick and 
Horstmann, and later by Howe, et al. (1950), had shown that intestinal infection was ac- 
companied by the appearance of neutralizing antibodies, and that the animals were re- 
sistant to subsequent challenge with the homologous virus type given by mouth. Subse- 
quently, studies conducted by Fox and associates in 1956 on families before and after 
episodes of natural infection with polioviruses indicated a similar pattern in humans. 
It seemed clear that if a safe oral vaccine could be developed, not only would individuals 
be immunized, but herd immunity could be achieved, so that circulation of wild polio- 
viruses could be blocked. This was an important consideration, for Sabin and others 
had demonstrated that although Salk vaccine induced serum antibodies which protected 
the individual against paralysis, it did not provide significant intestinal resistance and 
therefore was not so effective in preventing spread of the virus in a community. 

Sabin had made the important discovery that the CNS of monkeys was more sus- 
ceptible to polioviruses than that of higher primates (chimpanzees), and by inference, 
man. The reverse was true of the intestinal tract — man's being the nriost susceptible, 
that of chimpanzees and cynomolgus naonkeys next, but rhesus monkeys hardly at all. 
It remained, therefore, to identify nonneurotropic strains which would multiply exten- 
sively in the human intestinal tract and induce immunity without the risk of paralysis. 
Sabin therefore undertook an extensive series of experiments in which he was able to 
segregate avirulent strains of each of the three virus types by serial passage in 
cynomolgus -monkey kidney-tissue culture. Using both the terminal dilution method, 
and later the plaque technique introduced by Dulbecco and Vogt (1954), he obtained 
progeny from single poliovirus particles which provided optimal avirulent strains of the 
three types of polioviruses for use in attenuated vaccines. Koprowski and Cox of the 
Lederle laboratories also developed similar attenuated strains at this time (Paul, 1971). 
Eventually, however, exhaustive comparative tests for neurovirulence by intraspinal 
inoculation of monkeys gave the advantage to the Sabin strains. 

Sabin' s early studies involved many thousands of monkeys and hundreds of chim- 
panzees. Subsequently, when the attenuated strains were given to susceptible adult vol- 
unteers, it was found that they regularly induced a silent infection in the human intesti- 
nal tract as in chinnpanzees; virus excretion persisted for several weeks, and was ac- 
companied by the appearance of neutralizing antibodies in the serum, usually within 



L-11 

1 week. These observations were borne out in larger trials conducted among closed pop- 
ulations by Sabin and by others. In these studies it became apparent that the live-virus 
vaccines spread to some degree to exposed susceptible contacts and resulted in immuniz- 
ing infections in them as well. Cautious trials of increasing size in children in open 
communities nevertheless proved the safety and effectiveness of the attenuated vaccines — 
both those developed by Sabin and those developed by Koprowski and Cox. By 1957, the 
Lederle strains had been given to 250, 000 persons in the Belgian Congo; by 1959, the 
Sabin vaccine, prepared from seed lots provided by Dr. Sabin to the Soviet investigators, 
Chumakov and Voroshilova, and Smorodintsev, had been used to immunize some 15 mil- 
lion persons in the USSR and other countries of the Soviet bloc (Paul, 1971). These 
rapid developments were followed by the convening of two international conferences held 
in Washington, D. C. , in 1959 and in 1960, under the sponsorship of Pan American Sani- 
tary Bureau (now the Pan American Health Organization), a division of the World Health 
Organization. The meetings were financed not by the NFIP, but by the rival organiza- 
tion, the Sister Elizabeth Kenny Foundation of Minnesota. An enormous amount of new 
information was presented by workers from many countries. Clearly, live attenuated 
vaccines had arrived. In 1960-1961 the Sabin strains were licensed for use in the U. S. 
Unfortunately, as was the case folio-wing licensure of the Salk vaccine, all -was not 
smooth sailing thereafter. A certain number of probable vaccine-associated cases in 
vaccines and their contacts appeared in the U. S. in the first years of use; these, how- 
ever, have diminished steadily and had become very rare by 1975. 

During the years from 1960 on-ward, the impact of the live-virus vaccine resulted 
in a further decline in incidence of poliomyelitis in many parts of the world. The total 
number of cases reported in the U. S. fell from an average of 21, 000 per year from 
1950-55, to 7 in 1974 — an incredible record. While Salk vaccine continued to be used 
in the early 1960's, it was gradually supplanted in the U. S. by the oral vaccine, which 
is now the only one available. The reasons for this shift relate to several features of 
the live-virus vaccine: its superior immunogenicity, which results in lasting intestinal 
resistance; the speed with which high levels of antibody are achieved; and the logistic 
simplicity of an oral preparation which obviates multiple injections and the necessity for 
repeated revaccination. That the Salk-type vaccine can also achieve remarkable long- 
term freedom from poliomyelitis is nevertheless illustrated by the experience in Sweden, 
where it has been the only one used. The environmental conditions in this small coun- 
try with a high standard of living, a homogeneous population, close public health sur- 
veillance, and control of immunization practices account for this success. Yet for most 
countries, particularly the developing ones, oral vaccine has remained the method of 
choice for the prevention of paralytic poliomyelitis. 



REFERENCES 

Anderson, F. J., and Frost, W. H. , J. Amer. Med. Ass., 5_6, 633 (1911). 

Armstrong, C. , Amer. J. Publ. Hlth. , 27, 103(1937). 

Armstrong, C. , Publ. Hlth. Rep. (Wash.), 54, 17 19(1939), 

Armstrong, C. , Publ. Hlth. Rep. (Wash.), 54, 2302 (1939). 

Armstrong, C. , and Harrison, W. T. , Publ. Hlth. Rep. (Wash.), 50, 725(1935). 



L-12 

Bodian, D. , Proc. Soc. Exptl. Biol. Med., 72, 259(1949). 

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Brodie, M. , and Park, W. H. , N. Y. St. J. Med., 35, 815(1935); J. Amer. Med. 
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Burnett, F. M. , and Macnamara, J., Brit. J. Exp. Path., U^, 57(193 1). 

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Clifton, C. E. , Introduction to the Bacteria, 1st ed. , McGraw-Hill Book Co., Inc., 
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Hyg., 54, 191 (1951). 

Draper, G., Acute Poliomyelitis, P. Blakiston's, Philadelphia, 8 (1917). 

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Enders, J. F. , Weller, T. H. , and Robbins, F. C. , Science, 109 , 85(1949). 

Enders, J. F. , Weller, T, H. , and Robbins, F. C. , Fed. Proc, j_l_, 467(1952). 

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Flexner, S., and Lewis, P. A., J. Amer. Med. Ass., 54, 1780 (1910). 

Fox, J. P., et al. , Amer. J. Publ Hlth. , 46, 283 (1956). 

Frost, W. H. , Hyg. Lab. - Bull., No. 90 (1913). 

Harrison, R. G. , Proc. Soc. Exp. Biol. Med. (N. Y. ), 4, 140 (1906-1907). 

Heine, J. V., Spinale Kinderlahmung, Cotta, Stuttgart (1860). 

Horstmann, D. M. , Proc. Soc. Exp. Biol. Med. (N. Y. ), 79, 417(1952). 

Horstmann, D. M. , McCallum, R. W. , and Mascola, A. D., J. Exp. Med., 99, 355 
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Howe, A. H. , and Bodian, D. , Bull. Johns Hopkins Hospital, 69, 149 (1941). 

Howe, H. A., Bodian, D. , and Morgan, I. M. , Amer. J. Hyg., 5J_, 85(1950). 

Kling, C. , Acta Soc. Med. Suecanae, 55, 1 (1929). 

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Stockholm, 3, 5 (1912). 

Kolmer, A. J., J. Amer. Med. Ass., 105 , 1956(1935). 

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L-13 



Landsteiner, K. , Levaditi, C. , and Pastia, C. , Ann. Inst. Pasteur, 25, 805 (1911). 

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Melnick, J. L. , and Horstmann, D. , J. Exp. Med., 85, 287(1946). 

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Paul, J. R. , Riordan, J. T. , and Melnick, J. L. , Amer. J. Hyg. , 54, 275(1951). 

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L-14 

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Tisdall, F. F. , Brown, A., deFries, R. D. , Ross, M. A., and Sellers, A. H. , 
Canad. Publ. Health J. , 2_8, 523 (1937). 

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Phycns. , 52_, 306 (1937). 

Trask, J. D. , Vignec, A. J., and Paul, J. R. , JAMA, 111 , 6(1938). 

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3_8, 147 (1938). 

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Wickman, I., Beitrage zur Kenntnis der Heine-Medinschen Krankheit (Poliomyelitis 
Acuta und Verwandter Erkrankungen), Karger, Berlin ( 1907). 

Younger, J. S. , Ward, E. N. , and Salk, J. E. , Amer. J. Hyg. , 55, 301 (1952). 



DISCOVERY OF VIRUS AND PATHOGENESIS 



1908 Landstejner Discovery of the vin 
poliomyelJlis 
MR/D/W/P/M 



1910 Flexner Neutralizing antibodies to 
poliovirus found in experi- 
mentally infected monkeys 



DISCOVERY OF VIRUS AND PATHOGENESIS 



TISSUE CULTURE OF POLIOUIRus 



n NnitttlUIng tnllbDitll Fo 
(Bltofnytlilli and li norir 
MR/S/U/G/GL 



ANTIGENIC TYPING OF POLIOVIRUS 



INACTIVATED VACCINE 



ATTENUATED VACCINE 



MftlSIUIP.CfM 



MR/S/U/P/M 



mh/s/u/p;m 



unionimi 






..™,,J 



CODES 

NH Nonmiuion-OilenleJ Hcword 

MR Mtisioo-Oiteiited Beicaidi 

OV Osvtlopfneni 

S Saniliuni E.efH 

D Dcciiivn E.cnl 

U U. S. Ev«<n 

W Fofilgn E>enl 

G Govcrnmni Fundmfl 

p pfi.d<e Fundlna 

M Medical School - HoipiMI - 

1 Rewi'cii Initlluilcn 

OL GowrnmBnt Lifaomofv 

C CofWBny 



MR/S/UfP/M 



MR«/U/P/M 






HISTORIOGRAPH - POLIO VACCINE 



:\ 



M-1 
APPENDIX M. RH ANTISERUM ("VACCINE") 

INTRODUCTION AND SUMMARY 



The conquest of Rh disease through the elucidation of the disease, development 
of effective diagnostic and therapeutic procedures, and eventual prophylaxis of the 
disease represents one of the major medical achievements of this century. The major 
portion of this progress occurred over a 30-year period (i.e., 1936-1968). This is 
a remarkably short period of time to perform the quantity and quality of research 
generally required to gain an understanding of any complex disease process and 
proceed to treat and/or prevent the disease. 

The Rh "vaccine" (hyperimmune anti-D [ Rho] gamma globulin) has been clinically 
so effective in preventing Rh sensitization that experts in the area believe that Rh 
disease will cease to be a problem in this generation. There are of course small 
numbers of presensitized females, and transfusion accidents due to human error will 
occur, but generally speaking Rh disease has been effectively controlled. 

The successful control of Rh disease is the result of the merging of several 
separate areas of research. The description of the etiology of erythroblastosis fetalis 
and the histopathological characterization of hemolytic diseases of the newborn are 
the two main historical lines of early research. During this same time period (roughly 
1900 to 1950) there were parallel lines of methods of therapy and of detection which 
were generally impractical for the large number of affected individuals. Both the 
early bilirubin detection methods and the subsequent therapeutic intrauterine trans- 
fusions were effective only in those cases in which the disease could be rapidly diag- 
nosed and were useless as a means of prophylaxis. 

Three major time clusters of experimentation occurred [N.B. — Historiograph, 
i.e., (1) Characterization, 1 913- 1 932; (2) Etiology, 1 938- 1943; and (3) Etiology, 1954- 
1958] which led investigators to the final correct approach to prophylaxis via adminis- 
tration of Rh antiserum (1956-1968). There were a number of motivating forces 
behind the eventual development of the "vaccine". First, there was the 20-year 
publication rivalry between Weiner and Levine which provided the impetus to keep 
both of these investigators on relatively restricted (and generally correct) lines of 
research. Second, there were the extensive trials of preliminary "vaccines" by Freda 
in this country and Finn and Clarke in Liverpool, England. Third, there was the 
financial support supplied by Ortho Diagnostics to implement a usable Rh antiserum. 
Fourth, there was the organization of the Working Party on the Use of Anti-D Immuno- 
globulin in England. 

At present the early diagnosis of erythroblastosis fetalis can be followed by 
early delivery, intrauterine transfusion, or exchange transfusion of the newborn. 
These techniques are necessary only in those women who have been presensitized or 
who have not received the Rh vaccine after the first pregnancy. For those women who 
do receive the Rh vaccine upon the completion of their first pregnancy, the problem of 
isoimmunization to their fetus and to subsequent fetuses is almost completely 
eliminated. 



M-2 
HISTORICAL ACCOUNT 

Characterization of Erythroblastosis Fetalis 



Although hemolytic disease of the newborn was described prior to the turn of 
this century, the relationship between jaundice (icterus), hydrops (swelling), anemia, 
and eventually brain damage (kernicterus) in newborns was not established until the 
early 1900's (Hirch, 19l3;Yllpo, 1 9 13; Goldbloom and Gottlieb, 1929; Ferguson, 1931). 
These early reports were primarily disease descriptions based on fetal autopsies. In 
1932, Diamond et al. demonstrated that erythroblastosis was the single disease under- 
lying jaundice, swelling, and anemia. Following this descriptive phase, a relationship 
was established between the level of bilirubin in the infant's serum and kernicterus 
(brain damage) and methods of bilirubin quantification were developed (Weech, 1947; 
Vaughn etal., 1950; Allen et al. , 1950; Hsia et al. , 1952). Subsequent to these 
developments, the presence of hyperbilirubinemia was shown by Hsia in 1952 to be 
directly related to the delayed brain damage (kernicterus) noted in infants with erythro- 
blastosis fetalis. 



Etiology of Erythroblastosis Fetalis 



Concurrent with disease interrelationships and their description, the major blood 
groups (A, B, and O) were recognized by Lansteiner in 1900. Several additional 
blood group isoantigens were identified by Landsteiner and Levine in 1927 and Levine 
and Stetson noted the presence of a non-ABO blood group antibody in the serum of a 
patient following delivery of a stillborn infant in 1939. The following year, Landsteiner 
and Weiner observed that an antiserum prepared in rabbits against an antigen on the 
erythrocytes of the rhesus monkey reacted positively with 85 percent of human erythro- 
cytes, which presumably contained the antigen, while 15 percent of those tested were 
negative. This non-ABO blood group antigen, subsequently shown to be a system of 
antigens, was termed the Rh antigen. 

In 1941 Levine et al. demonstrated that Rh sensitization in an Rh-negative mother 
to an Rh-positive fetus was responsible for the disease pathologies associated with 
erythroblastosis fetalis. The protection against Rh sensitization afforded by ABO in- 
compatibility between the mother and fetus was postulated by Levine in 1943 and sub- 
sequently substantiated by statistical analysis also prepared by Levine and reported 
in 1958. 



Disease Detection 



In the 1950's several procedures developed for the detection of both fetal erythro- 
cytes and/or antibodies to Rh-positive fetal erythrocytes in the maternal circulation 
proved the relationship between Rh isoimmunization and erythroblastosis fetalis. 
Fetal hemorrhage into the maternal circulation was documented by Chown in 1954, who 
demonstrated the presence of agglutinins to an Rh-positive fetus in an Rh-negative 
mother. Placental transfer of fetal erythrocytes was shown by Mengert and coworkers 
the following year (1955), and a test was developed by Kleihauer and his associates in 
1957 to demonstrate the presence of fetal erythrocytes in the maternal circulation. 



M-3 

Several approaches to earLy disease detection were developed folLowing elucida- 
tion of the etiology of Rh hemolytic disease. The earliest of these tests involved testing 
the mother's serum for the presence of incomplete Rh antibodies (Coombs et al. , 1945, 
Diamond and Abelson, 1945), A second approach, initially developed by Van der Bergh 
and Muller in 1916 and refined by Hsia et al. in 1952, was based on the detection of 
bilirubin in the serum of the neonate and somewhat later applied to the amniotic fluid 
of the mother (Bevis, 1956). The establishment of the relationship between bilirubin 
levels and brain damage in Rh hemolytic disease led Hsia and coworkers in 1952 to 
perfection of a micromethod for determination of bilirubin levels in the serum of 
diseased infants. This assay provided a basis for the development by Diamond et al. 
in 1951 of successful umbilical exchange transfusions in the affected neonate. The 
development by Bevis in 1956 of transabdominal amniocentesis for the purpose of 
determination of bilirubin levels in the amniotic fluid (spectrophotometric analysis) 
enabled Liley to monitor more precisely the extent of hemolytic disease in the fetus and 
Led eventually to Liley's highly successful treatment in 1963 of the diseased fetus in utero 
by intrauterine transfusion. 

Another approach to detection of maternal Rh sensitization against incompatible 
fetal erythrocytes involved the demonstration of fetal cells in maternal blood. This 
technique, introduced by Kleihauer et al. in 1957, has been widely applied to monitor 
the presence of fetal red cells in the maternal circulation. It remains the major 
method of demonstrating that a transplacental hemorrhage of fetal erythrocytes into 
the maternal circulation has occurred. 



Therapy of Erythroblastosis Fetalis 

As noted in the previous section, the therapy of Rh hemolytic disease was directly 
dependent on the perfection of accurate diagnostic procedures and elucidation of the 
mechanism of the disease process and the resultant pathologic manifestations. 

The earliest surgical approach to disease treatment was exchange transfusion of 
the affected neonate shortly after birth. The first successful exchange transfusion was 
performed by Hart in 1925 for the disease then termed icterus gravis. In the 1940's 
a great deal of experimentation was performed to perfect this procedure for the early 
treatment of neonates with previously detected erythroblastosis fetalis, Wiener re- 
ported in 1948 on several successful exchange transfusions via the antecubital vein in 
infants with erythroblastosis fetalis. In 1951 Diamond et al. reported on the further 
refinement of this procedure using the more accessible umbilical vein and a clot- 
retarding plastic catheter. Diamond has stated that the extension of this method to 
multiple transfusions in the diseased live-newborn virtually eliminated the threat of 
brain damage, and the mortality of neonates with erythroblastosis dropped to about 
2. 5 percent. 

However, the problem of the severely diseased fetus which usually died in utero 
required a more sophisticated therapeutic approach. This advance became possible 
following the perfection of amniocentesis to monitor accurately bilirubin levels in the 
gravid sensitized female. Approximately 25 percent of Rh-positive fetuses in Rh- 
sensitized females were destined to be stillborn. The intrauterine transfusion intro- 
duced by Liley in 1963 was directly responsible for eventually preventing stillbirth 
in more than 60 percent of these cases, Adamsons et al, attempted without success 
to refine this procedure further via hysterotomy in 1965, The only additional 



M-4 

therapeutic technique which has been applied to erythroblastosis fetalis is phototherapy 
(Lucey et al. , 1968). This type of therapy is used as an adjunct to exchange transfusion 
to control rising bilirubin levels in diseased neonates. 



Prophylaxis of E rythroblastosis Fetalis 



Rh sensitization may occur following the delivery of the first Rh-positive infant 
to an Rh-negative female. Sensitization may also occur because of a slow transfer of 
fetal erythrocytes across the placenta during pregnancy, following an abortion, or may 
even be the result of administering Rh-positive donor blood to an Rh-negative recipient. 
Mechanistically, the sensitization process involves the formation of specific Rh{D) 
(both Rh and Rh-D used to identify the Rh antigen) antibody in the female to the Rh anti- 
gen on the fetal erythrocyte. In a sensitized female, the 7S immunoglobulin can cross 
the placenta and damage fetal red cells. The formation of Rh antibody in the sensitized 
female is termed an anamnestic response. The current protocol employed to prevent 
Rh sensitization is based on the observation by Smith in 1909 that the presence of 
excess passive antibody prevented active immunization to the corresponding specific 
antigen. 

The natural protection afforded the fetus in the ABO-incompatible situation led 
several investigators to speculate on the possibility of simulating this protection by 
administering serum containing Rh antibodies. The earliest proposal that passively 
administered Rh antibody at delivery might prevent sensitization apparently was made 
by Finn and his associates in Liverpool. Two groups of investigators (Finn et al. , 
1961 and Freda and Gorman, 1962) administred Rh antiserum to Rh-negative male 
volunteers, with the same conclusion — passively administered Rh hyperimmune anti-D 
(Rho) gamma globulin could prevent sensitization. 

The trials of the Rh antiserum in women at risk following delivery of a first Rh- 
positive neonate began in New York and in Liverpool in 1964. In 1966 Freda et al. 
demonstrated in a statistically significant study protection of Rh-negative women 
following an Rh-positive birth by administration of Rh antibody prepared from concen- 
trated gamma globulin. The positive results of Clarke et al. reported in 1967 (Liver- 
pool Group) are perhaps more dramatic owing to the almost exclusive use of high-risk 
individuals in their clinical trials. 

During this period the Rh vaccine (RhoGAM, as it was termed by Ortho) was , 
being developed and was made commercially available in 1968. The "vaccine", 7S-anti- 
Rh antibody, was prepared from the plasma or serum of Rh-sensitized, Rh-negative 
individuals . 

The concept that specific Rh antibody mediated suppression of immunization by the 
Rh antigen was thus demonstrated and the "vaccine" was miade available for general use 
by 1968. This approach to prophylaxis has since been shown to be nearly 100 percent 
effective by following high-risk, Rh-negative women through successful second preg- 
nancies, and minimum prophylactic doses have been established through extensive 
clinical trials (Working Party on the Use of Anti-D-Immunoglobulin for the Prevention 
of Isoimmunization of Rh-Negative Women During Pregnancy, 1974). 



M-5 

REFERENCES 

Adamsons, S. K. , Freda, V. J., et al. , Pediat. , 3_5, 848(1965). 

Allen, F., Diamond, L. , et al. , Amer. J. Dis. Child., _80, 779(1950). 

Bevis, D.C.A., J. Obstet. Gynaec. Brit. Comm. , _63, 68(1956). 

Billings, B. H. , and Lathe, G. H. , Amer. J. Med., 24, 111 (1958). 

Charles, A. G. (ed.), Rh Isoimmunization and Erythroblastosis Fetalis, Appleton- 
Century Crafts, New York ( 1 969) . 

Chown, B., Lancet, 1^, 1213 (1954). 

Clarke, C. A., et al. , Brit. Med. J., 4, 7 (1967). 

Coombs, R.R.A., Mourant, A. E., et al. , Lancet, 2, 15 (1945). 

Diamond, L. K. - Interview. 

Diamond, L. K. , Amer, J. Clin. Path., _62, 311 (1974). 

Diamond, L. K. , and Abelson, N. M., J. Lab. Clin. Med., 3^, 204(1945). 

Diamond, L. K. , Allen, F. H. , et al. , N. Eng. J. Med., 244, 39 (1951). 

Diamond, L. K., Blackfan, R. C, et al. , J. Pediat. , J., 269(1932). 

Ferguson, J. A., Amer. J. Path., 7, 277(1931). 

Finn, R. , Clarke, C. A., et al. , Brit. Med. J., 1, 1486 (1961). 

Freda, V. J., and Gorman, J. G. , Bull. Sloane Hosp. Women, 8, 147(1962). 

Freda, V. J., and Gorman, J. G. , Science, 151, 828 (1966). 

Gold, E. R. , and Butler, N. R., ABO Haemolytic Disease of the Newborn, The 
Williams and Wilkins Company, Baltimore (1972), 

Goldbloom, A., and Gottlieb, R., Amer. J. Dis. Child., 3_8, 57 (1929). 

Hart, A. P., Can. Med. Assoc. J., V5, 1008(1925). 

Hirsch, A., Z. Kinderheilk . , 9, 196(1913). 

Hsia, D.Y.Y., Hsia, H. H. , etal., J. Lab. Clin. Med., 40, 610(1952). 

Hsia, D. Y., Allen, F. H. , et al. , N. Eng. J. Med., 247, 668 (1952). 

Kleihauer, E., Braun, H. , and Betke, K., Klin. Wschr, , 35, 637 (1957). 

Klieger, J. A., Med. Clin. N. Am., _53, 1063 (1969). 

Landsteiner, K. , Zbl. Bakt. , 27, 357(1900). 

Landsteiner, K. , and Levine, P., Proc. Soc. Exp. Biol. Med., 24, 939 (1927). 

Landsteiner, K. , and Levine, P., Proc. Soc. Exp. Biol. Med., 24, 600 (1927). 

Landsteiner, K., and Wiener, A., Proc. Soc. Exp. Biol. Med., A3_, 223 (1940). 

Levine, P., Hum. Biol., 3^, 14(1958). 

Levine, P., Katzin, E., etal., JAMA, 116, 824(1941). 



M-6 

Levine, P., and Stetson, R. , JAMA, _n3, 126 (1939). 

Levine, P., J. Hered. , 34, 71 (1943). 

Liley, A. W., Brit. Med. J., 2, 1107 (1963). 

Lucey, J. F. , Ferreird, M. , et al. , Pediat. , 41, 1047(1968). 

Mengert, W. F. , Rights, G. S., et al. , Amer. J. Obstet. Gynec, _69, 678(1955). 

Smith, T., J, Exp. Med. Biol., U_, 241 (1909). 

Stern, K., Davidsohn, M. D., et al. , Amer. J. Clin. Pathol., 26, 833(1956). 

Van der Bergh, A. A. H. , and MuUer, P., Biochem. Z., T7, 90(1916). 

Vaughn, V. C, Allen, F. H., Jr., et al. , Pediat., 6, 706(1950). 

Weech, A. A., Advances in Pediat., 2, 346 (1947). 

Weiner, W. , Child, R. M., etal., Brit. Med. J., 2, 770(1958). 

Wiener, A. S,, J. Lab. Clin. Med., 3J., 1016(1945). 

Wiener, A. S., and Peters, H. R. , Ann. Intern. Med., 13, 2306 (1940). 

Working Party on the Use of Anti-D-Immunoglobulin for the Prevention of Isoimmuni- 
zation of Rh-Negative Women During Pregnancy, Brit. Med. J., 2 (910), 75(1974). 

YUpo, A., Z. Kinderheilk., 9, 208(1913). 

Zimmerman, D. R. , Rh. The Intimate History of a Disease and Its Conquest, 
Macmillan Publishing Company, Inc., New York (1973). 



CHAHACTEHrZATION OF HEMOLYTIC DISEASE OF THE NEWBORN 
(ERYTHROBLASTOSIS FETALIS! 



ETIOLOGY OF ERYTHROBLASTOSIS FETALIS 



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HISTORIOGRAPH - Rh ANTISERUM ("VACCINE") 



N-1 
APPENDIX N. RUBELLA VACCINE 

INTRODUCTION AND SUMMARY 



Rubella in children and adults is generally characterized as a mild exanthem 
which rarely produces complications. However, Selzer's demonstration in 1963 that 
rubella virus is the definitive etiological agent of the congenital rubella syndrome 
(CRS) provided irapetus to develop a vaccine. 

In discussiag the medical significance of congenital rubella, several points need 
to be made. 

(1) Congenital rubella is a disease that involves many body organs and 
results in a wide range of defects. The physical findings are often 
accompanied by a variety of behavioral manifestations. For 
proper management of the multihandicapped rubella child, facilities 
are required that go beyond just the deaf or deaf- blind child for 
which they are currently designed. Facilities are needed to deal 
with the multiple problems that may arise with certain children. 

(2) Congenital rubella is not only a multisystem disease, but a con- 
tinuing disease. Therefore, long-term evaluations are necessary 
so that defects not evident at birth are not overlooked and so that 
the etiology of later problems can be identified. 

(3) Mental retardation is a major consequence of congenital rubella. 
In 1971, Chess et al. reported that 37 percent of infected children 
had varying degrees of retardation. This figure is remarkable 
when one considers that the expected prevalence in the general 
population is 2 to 3 percent. 

It is clear that congenital rubella has a profound effect both on the affected child 
and the family. The need, therefore, to protect the pregnant female from infection 
by the use of appropriate vaccines is pressing. To this end, rubella vaccines have 
been available since 1969, and what follows is a historical account of vaccine develop- 
ment and a discussion of the effectiveness of the vaccines. 



HISTORICAL ACCOUNT 



Clinical Studies of Rubella and the Congenital Rubella Syndrome 



Rubella was first described as a disease entity by de Bergen in 1752. The disease 
was reported to be distinct from measles by Veale in 1866. Rubella in children and 
adults is generally characterized as mild exanthem which rarely produces complications. 
However, as the result of an epidemic of rubella in Australia in 1939-1940, an unusual 
number of cases of congenital cataract were observed from areas throughout Australia. 
These findings prompted Norman Gregg in 1941 to conduct retrospective studies which 
associated for the first time maternal rubella with congenital malformations. 



N-2 

Shortly after Gregg's report two large-scale investigations were conducted in 
Australia. Swan et al. in 1943 studied congenital defects in neonates in southern Aus- 
tralia; in New South Wales a committee was appointed by the Director General of 
Health to investigate Gregg's observations. These investigations confirmed and ex- 
panded the original findings. The major congenital defects found were deaf-mutism, 
eye and heart disease, and possible mental defectiveness. In addition, there was evi- 
dence that the type of defect was related to the time when maternal rubella occurred; 
the highest risk was in the first trimester of pregnancy. 

Following the original reports by the Australians, observers in other countries 
reported similar findings. Investigators approached the subject from different view- 
points according to their own specialities. Carruthers (1945) reported on severe deaf- 
ness in the neonate. Dogramaci and Green (1947) described congenital heart disease. 
In 1949, Kamerbee reported on progressive deafness in the neonate, and Mutrus (1949) 
observed retarded myelinization as an effect of congenital rubella. Other investigators 
have studied the short-term and prolonged effects of congenital rubella in regard to 
temporal bone involvement, cataracts, and neurological damage. 

The United States epidemic of rubella in 1963-65 confirmed and expanded on the 
defects observed previously. Rudolph et al. in 1965 described heart and eye defects. 
Severe neurological disturbance was identified by Desmond et al. in 1967, and in 1973 
Rorke described ischemic brain damage resulting from congenital rubella. 



Isolation, Identification, and Association of Rubella 
With Congenital Rubella Syndrome 



The focus on rubella began with Gregg's observations in 1941 associatingmaternal 
rubella with congenital abnormalities. The development of laboratory techniques per- 
mitting specific diagnosis of rubella, however, did not occur until some 20 years later. 
The rubella virus was isolated in 1962, independently, by Weller and Neva using human 
amnion cell culture, and Parkman and associates using African green monkey kidney 
cells. Subsequently, in vitro studies by Plotkin in 1965 and Rawles in 1966 have shown 
that rubella- virus-infected cells undergo mitotic inhibition and chromosome breakage. 

Selzer in 1963 and Alford et al. in 1964 were successful in isolating rubella 
virus from human fetal tissue, demonstrating for the first time a definitive etiologic 
agent of the congenital rubella syndrome. Monif and Sever in 1965 reported that rubella 
virus could be isolated from a variety of clinical specimens, including throat swabs, 
rectal swabs, cerebrospinal fluid, liver biopsy, and urine. In addition, several inves- 
tigators have isolated rubella virus from the lens of the eye of congenitally infected 
fetuses. Hambridge, et al. in 1966 reported that infants with congenital rubella syn- 
drome excrete rubella virus in the urine for many months after birth, creating a poten- 
tial source of contact infection. 

Following the isolation of rubella virus in cell cultures, serological techniques 
to detect immunity to rubella were rapidly developed. Parkman et al. in 1964 de- 
scribed a virus neutralization test for detection of serum antibody. It was shown that 
following infection with rubella virus, neutralizing antibodies developed which persisted 
indefinitely, conveying a high order of protection against reinfection. Other diagnostic 
serological techniques also have been described. Brown et al. in 1964 demonstrated 
that antibodies to rubella virus could be detected by the immunofluorescence test. 



N-3 

Sever et al. in 1965 described a complement-fixation test for detection of immunity to 
rubella, A most significant finding was reported by Stewart et al. in 1967 that rubella 
virus would agglutinate erythrocytes (hemagglutination) and that antibody to the virus 
would inhibit hemagglutination. As with neutralizing antibodies, hemagglutination- 
inhibition (HAI) antibodies appear at the end of the first week after symptoms of rubella, 
reaching peak levels 10 to 21 days after onset, and persist indefinitely. Consequently, 
for diagnostic purposes, an acute serum sample is obtained at onset of symptoms, 
and after 2 weeks a convalescent serum sample is obtained. A fourfold rise in antibody 
titer is diagnostic. During the years 1967-69, several independent investigators con- 
ducted comparative studies of diagnostic techniques for detection of rubella. As a 
result, the HAI test has largely replaced the neutralization, complement fixation, and 
immunofluorescence tests for determining immunity status to rubella. 

In 1966 Tondury reported that the placenta plays a role in maternal-fetal trans- 
mission of rubella. These observations were confirmed by Singer in 1967 and extended 
in 1968 by Hancock, who described the development of antibodies of the IgM type in 
the newborn resulting from in utero infection. In considering the diagnostic implication 
of this finding, it is significant to note that antibodies of maternal origin which cross 
the placenta are of the IgG type and the IgM antibody is of fetal origin. Both IgG and 
IgM antibodies can be measured by the HAI test. During the early postnatal months, 
the transplacentally acquired IgG is lost; however, IgM production is continued. 
Therefore, the persistence of HAI antibodies (IgM) through the first year of life sup- 
ports the diagnosis of congenital rubella. 

A significant refinement of the HAI test was described in 1969 by Cooper et al. 
They found that heparin-manganese chloride treatment of sera would remove nonspecific 
inhibitors of rubella hemagglutination, allowing a reliable detection of HAI antibodies. 

In 1970, the Center for Disease Control (CDC) recognized the need for standard- 
izing the rubella HAI test, and consequently formulated a standard protocol for the 
performance of the test based upon comparative studies among several laboratories. 



Development of Vaccines 



Following the isolation of rubella virus, impetus was provided to develop a 
vaccine. Since rubella is generally a mild illness, the principal objective of the vac- 
cine is to prevent infection of the fetus and the resulting congenital rubella syndrome. 

In 1966, Parkman, Meyer, and associates attenuated rubella virus by 77 serial 
passages in primary African green monkey kidney (GMK) cell culture (HPV-77). The 
first clinical vaccine trials were by Meyer et al. in 1966, using HPV-77 rubella strain 
as a vaccine. Subsequently, other attenuated rubella strains have been derived from 
HPV-77, among which are HPV-77 passaged five times in duck embryo fibroblast cell 
culture (HPV-77DEr) and HPV-77 passaged 12 times in dog kidney cell culture 
(HPV-77DK22) . In^ addition, an attenuated rubella virus strain (Cendehill) passaged 
51 times in primary rabbit kidney cell culture has been developed. 

Experimental animal models for studies of rubella have not been particularly re- 
warding, although Parkraan et al. demonstrated in 1965 that congenital infection can 
be induced in the rhesus monkey. This finding was confirmed by Delahunt and Rieser 
in 1967. Prior to licensing of live, attenuated rubella vaccine in 1969, studies were 



N-4 

conducted in the rhesus monkey. The findings were that the vaccine virus did not 
cross the placenta. 

Subsequent vaccine trials using HPV-77 rubella strain derivatives and Cendehill 
strain have demonstrated that attenuated rubella vaccine does confer seroconversion 
in approximately 95 percent of vaccines. However, it is nov/ apparent that, contrary 
to the findings in rhesus monkeys, attenuated rubella vaccine virus can cross the 
placenta and infect the human fetus. Vaccine virus has been recovered from fetal 
tissue after accidental vaccination of pregnant women, reported by Phillips et al. in 
1970, and after purposeful inoculation of vaccine virus in women about to undergo 
planned abortions, as described by Vaheri et al. in 1969. 

In 1971 the United States Public Health Service initiated a "Herd Immunity" pro- 
gram. Because, in an epidemiological sense, children represent the major "herd" 
of susceptibles that the virus requires to maintain itself, a general inoculation of 
children ranging in age from 1 year to puberty was initiated. However, outbreaks con- 
tinued with cases in other unvaccinated age groups. An epidemic occurred in 1971 in 
Casper, Wyoming, involving 1, 039 persons primarily in two high schools and three 
junior high schools. These findings have prompted several authors to propose that 
the concept of herd immunity is invalid, and that outbreaks among adolescents and 
adults demonstrate the inadequacy of childhood vaccination. 



REFERENCES 



Alford, C. A,, Jr., Neva, F. A., et al. , New Eng. J. Med., 27J., 127 5 (1964). 

American Academy of Pediatrics, Report of the Committee on Infectious Diseases, Ed. 
17, Evanston, Illinois, AAP(1974). 

Bolognese, R. J., and Carson, S. L. , Obstet. Gynecol., 42, 851 (1973). 

Bolognese, R. J., Carson, S. L. , et al. , Am. J. Obstet. Gynec. , 112 , 903 (1972). 

Brown, G. C, Maassab, H. F. , et al. , Science, j^, 943 (1964). 

Carruthers, D. G. , Med. J. Aust. , J,, 315 (1945). 

Center for Disease Control, U. S. Department of Health, Education, and Welfare, 
Public Health Service, Center for Disease Control, Atlanta, Ga. (1970). 

Center for Disease Control, Kansas City, Missouri and Atlanta, Ga. , The Center, 1 
(1971). 

Chess, S., Korn, S. T., et al. , Psychiatric Disorders of Children with Congenital 
Rubella , p. 147, Pub. Brunner /Mazel, Inc. (1971). 

Cooper, L. Z., Matters, B. et al. , J. Amer. Med. Ass., 207 , 89 (1969). 

Cotlier, E., and Fox, J., in Current Concepts in Ophthalmology, B. Becker and C. R. 
Drews (eds.), St. Louis, Mosby (1967). 

de Bergen, New Eng. J. Med., 236 , 943 (1947). 

Deibel, R. S., Cohen, M. , et al. , N. Y. State J. Med., _68, 1355 (1968). 

Delahunt, C. S., and Rieser, N., Am. J. Obstet. Gynec, 99, 580 (1967). 

Desmond, M. M. , Wilson, G. S., et al. , J. Pediat. , 71, 311 (1967). 



N-5 

Dogramaci, I., and Green, H. , J. Pediat. , 30, 295(1947). 

Ebbin, A. J., Wilson, M. G., et al. , Am. J. Obstet. Gynec. , JJ/7, 505 (1973). 

Field, A. M., Vandervelde, E. M. , et al. , Lancet, 2, 182(1967). 

Gregg, N. M. , Trans. Ophthal. Soc. Aust. , 3, 35 (1941).. 

Hambridge, K. M., Shaffer, D., et al. , Brit. Med. J., j., 650(1966). 

Hancock, M. P., Huntley, C. C., et al. , J. Pediat., 1Z_, 636(1968). 

Herrmann, K. L. , Halonen, P. E., et al, , Amer. J. Pub. Health, 59_, 296(1969). 

Kamberbeek, A.E.H.M., Verhandl van hit Instituuit por Praeventieve Geneskunde 14 
Leiden (1949). 

Karmody, C. S. , NewEng. J. Med., 2^8, 809 (1968). 

Katz, M., J. Pediatr., 84, 615 (1974). 

Klock, L. E., and Rachelefsky, G. S. , N. Eng. J. Med., 288, 69(1973). 

Korones, S. B., Ainger, L. E., et al. , J. Pediat., _67, 166(1965). 

Larson, H. E., Parkman, P. D., et al. , N. Eng. J. Med., 284, 870 (1971). 

Lennette, E. H. , Schmidt, N. J., et al. , J. Immunol., _99, 785(1967). 

Lindsay, J. R., and Harrison, R. S., J. Laryngol. Otolaryngol., 68, 461 (1954). 

Lundstrom, T. C., and Bloomquist, B. , Acta Pediat. , bQ_, 444(1961). 

McDonald, J. C., Brit. Med. J., 2, 416 (1963). 

Menser, M., Dods, L. , et al. , Lancet, 2, 1347(1967). 

Meyer, H. M. , Parkman, P. D., et al. , N. Eng. J. Med., _275, 575 (1966). 

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Murphy, A. M., et al. , Am. J. Ophthal., 64, 1109 (1967). 

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(1962). 

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Reid, R. R. , et al. , Med. J. Aust., _!., 540 (1966). 

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N-6 

Selzer, G. , Lancet, 2, 336(1963). 

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CHARACTERIZATION OF RUBELLA AND 
CONGENITAL RUBELLA SYNDROME 



ISOLATION, IDENTIFICATION. AND ASSOCIATION OF RUBELLA 
WITH CONGENITAL RUBELLA SYNDROME (CRS) 



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O-l 
APPENDIX O. PHENYLKETONURIA 

INTRODUCTION AND SUMMARY 



Sir Archibald Garrod's thesis oq "Inborn Errors of Metabolism" in 1908 provided 
a conceptual scheme of the relationship between gene, enzyme, and clinical abnormality. 
Since that time, more than 100 severe disorders resulting from a specific enzyme 
deficiency have been recognized. The least rare of these disorders is phenylketonuria 
(PKU). 

The incidence of phenylketonuria in the United States prior to blood-screening 
programs, was approximately 1:24,000 (Knox, 1966). This figure was derived primarily 
from screening retarded patients in mental institutions as well as from siblings identi- 
fied through urinary screening programs. More recent figures reported by Levy et al. 
(1970) and resulting from massive blood screening of newborn infants in Massachusetts 
place -the incidence at approximately 1:14,000. Most reports have indicated an equal 
incidence in male and female populations. However, recent preliminary data from the 
National Collaborative Study for Treatment of Phenylketonuria show an incidence ratio 
of 2: 1 for males to females (Hsia and Dobson, 1970). 

There are few symptoms of phenylketonuria in newborn infants. Vomiting and 
feeding problems may be apparent, and there is a high incidence of pyloric stenosis. 
Eventually, a peculiar odor associated with phenylketonuria is detected and infantile 
eczema may appear. The normal appearance continues for the first 1 to 3 months of 
life. Between 3 and 6 months, abnormal behavioral patterns become apparent and pro- 
pensive retardation occurs. Mental retardation is the one most detrimental defect in 
phenylketonuria. Mental retardation progresses to the extent that 65 percent of 
phenylketonurics possess an I.Q. equal to or less than 20 (Jervis, 1954). Over 90 per- 
cent of untreated phenylketonuria patients may be expected to have an I.Q. less than 
50 and eventually require institutionalization (Knox, 1972). Fortunately, untreated 
phenylketonuric children are seldom seen since massive screening of newborn infants 
and well-managed treatment programs have been initiated. Cost-benefit analysis 
has shown that such screening is beneficial not only to the diseased person but also to 
the general population (Stiner, 1973). The monetary savings to taxpayers when a suc- 
cessfully screened child is spared a lifetime admission to a state institution has been 
estimated at $100, 000 per child (Centerwall, et al., I960). Today, these savings 
should be substantially more. 

Phenylketonuria was discovered somewhat by accident in 1934 by a Norwegian 
doctor, Dr. Asbjorn Foiling, and became the first of the inherited disorders for which 
the postulates of Garrod were unequivocally demonstrated. In 5 months. Dr. Foiling 
published a report on a new metabolic disease that he postulated was an inherited 
disorder of phenylalanine metabolism. He called the disease "imbecillitas phenylpy- 
rouvica". Dr. Polling's diagnosis of a disease characterized by unusual phenylpyruvic 
acid loads in the urine associated with severe mental retardation was confirmed by 
Penrose in 1935. Subsequent work by Penrose (1935), Jervis (1939), and Munro (1941) 
showed that classical phenylketonuria, ascertained through mental retardation, was 
inherited as a simple autosomal mendelian recessive trait. The name phenylketonuria 
(PKU) was used by Penrose (1937) and remains the most commonly used term to de- 
scribe this metabolic disease. 



0-2 

Following the demonstration by Bernheim and Bernheim (1944) that the main 
metabolic pathway of phenylalanine was through its parahydroxylation to tyrosine, 
Udenfriend and Cooper (1952) described an enzyme system catalyzing the conversion 
of phenylalanine to tyrosine. One year later in 1953, Jervis showed that the liver of 
phenylketonurics was devoid of the phenylalanine-oxidizing enzyme. Subsequent re- 
search by Mitoma (1956), Wallace et al. (1957), and Kaufman and co-workers (1957- 
present) showed that the hydroxylating enzyme system is indeed a complex one requiring 
various protein cofactors and secondary enzyme systems. 

Cowie (1951) was the first to recognize "atypical phenylketonuria". Since that 
time, widespread screening has led to the recognition of cases of hyperphenylalanines 
which do not fit the definition of "classical" phenylketonuria. 

Prior to 1962, phenylketonuria was only sought in early infancy when the diagnosis 
had been made previously in a defective older sibling. Earlier in 1956, Armstrong and 
Binkley had observed that phenylketonuric infants appear biochemically normal at 
birth, and Armstrong and Binkley furthermore observed that urinary phenylpyruvic 
acid levels did not increase until 34 days after birth, while serum phenylalanine levels 
gradually increased from birth, reaching a maximum at 24 days. These studies indi- 
cated the need for early screening and for diagnosis based upon first, serum phenlya- 
lanine levels, and second, urinary levels of phenylpyruvic acid. 

Analysis of serum phenylalanine in phenylketonuria had been performed by Foiling, 
et al. , in 1938. However, the development of paper chromatography by Consden et al. 
(1944) and its subsequent application to the study of metabolic disorders by Berry in 
1951 provided an important event in the ultimate diagnosis of phenylketonuria and other 
metabolic disorders. 

Limited screening of infants for phenylketonuria in the late 1950's usually in- 
volved treating urine samples with ferric chloride. However, it was not until Guthrie 
reported on the microbial inhibition assay in 1961 that a simple, practical method was 
available for screening large populations for blood phenylalanine, thus permitting 
diagnosis of phenylketonuria before its symptoms became manifest. The first com- 
prehensive screening program began in Massachusetts in 1962 (see MacCready and 
Hussey, 1964). 

Presently, 45 of the 50 states in the United States require screening of newborn 
infants for PKU, The success of current screening programs and subsequent treatment 
has been documented in 1974 by Spencer and by MacCready, who observed no new admis- 
sions of phenylketonuria patients to mental institutions in Massachusetts since the intro- 
duction of massive screening in that state. 

Restriction of dietary phenylalanine is the only method to treat phenylketonurics. 
Phenylalanine-restricted dietary treatment of PKU was suggested by Woolf and Vulliamy 
in 1951. Armstrong, Woolf, and Bickel all initiated phenylalanine-restricted dietary 
regimens at about the same time. Credit for the first report on the effect of low- 
phenylalanine diets on lowering blood phenylalanine belongs to Bickel and co-workers 
(1953). Several reports on the effect of dietary treatment of phenylketonuria by Bickel, 
Woolf, Armstrong, Horner, and Berry followed. It soon became apparent that phenyla- 
lanine-restricted diets improved behavior, but mental damage could not be reversed. 
To be effective, treatment had to be started early in life or before the irreversible 
damage resulting from abnormal phenylalanine metabolism became apparent. Treat- 
ment by restriction of dietary phenylalanine became a somewhat emotional issue during 



0-3 

the late 1950's and 1960's. Researchers such as Bessman, Berman, and Birch 
questioned the effectiveness of dietary treatment. Others, including Woolf, noted 
gross overtreatment Leading to growth retardation primarily because of a lack of knowl- 
;edge of the nutritional requirements of the infant and sometimes incorrect diagnosis. 
In addition, questions pertaining to termination of dietary treatment have been raised. 
The problem of when and whether to terminate dietary treatment has not yet been re- 
solved. This and other problems are being considered in the National Collaborative 
Study on Treatment of Phenylketonuria started in 1967. Results from this study are 
still being collected. 



HISTORICAL ACCOUNT 



The historical account is divided into four sections, i.e., (1) discovery, descrip- 
tion, and etiology of phenylketonuria; (2) diagnosis and screening; (3) treatment; and 
(4) contributory events. For each section, key chronological events will be discussed. 
Somie of the events do not appear on the historiograph nor are all events on the historio- 
graph discussed in the text. The historiograph will illustrate the interrelationship of 
events from a chronological view. 



Discovery, Description, and Etiology 



In 1934, a Norwegian physician, Dr. Asbjorn Foiling, described 10 patients, 
several of them siblings, who excreted phenylpyruvic acid and were mentally deficient. 
The disease characterized by Foiling was the first of the inherited metabolic disorders 
for which the postulates of Garrod were unequivocally demonstrated. The story of the 
initial discovery is somewhat interesting and concerns an observant Norwegian mother 
with two handicapped children characterized by retardation or slowness in development 
and a very peculiar and clinging odor associated with the children. The children's 
father had been acquainted with Dr. Foiling in dental school and requested that Dr. 
Foiling examine the children. It was later discovered that Dr. Foiling was in fact 
related to the family in whom he discovered phenylketonuria. The initial discovery of 
phenylpyruvic acid in the urine of these children occurred somewhat by accident when 
the urine was examined for possible chronic infection by adding ferric chloride. Sur- 
prisingly, a distinctive green coloring appeared and was found to be a result of the 
reaction of ferric chloride with phenylpyruvic acid. It was hypothesized that this dis- 
order represented a disturbance in the metabolism of phenylalanine and the disorder 
was called "imbecillitas phenylpyrouvica". The diagnosis of Foiling was confirmed in 
1935 by Penrose. Subsequent studies by Penrose in 1935, Jervis in 1939, and Munro 
in 1941 showed beyond doubt that phenylketonuria ascertained through mental retarda- 
tion was inherited as a simple autosomal mendelian recessive trait. It was Penrose 
in 1937 who renamed the disease phenylketonuria. Continued studies by Foiling in 
1938 demonstrated that phenylalanine blood levels were higher in diseased patients and 
that phenylpyruvic acid levels were higher in heterozygotes than in normal patients. 
Foiling later performed phenylalanine loading experiments in attempts to show that 
high blood phenylalanine levels led to phenylpyruvic acid excretion. This resulted in 
excretion of phenylpyruvic acid in all persons tested. It was not known at this time that 
the preparation contained d-phenylalanine. It was later found that the d form always 



0-4 

Leads to phenyLpyruvic acid excretion. Because of the difficulty in obtaining sufficient 
amounts of L-phenyLaLanine, successful loading experiments were not done until many 
years later. It was significant during these early years that the disease was easily 
identified by reacting ferric chloride over the urine of phenylpyruvic patients. It was 
necessary only to apply the simple ferric chloride test to populations in institutions for 
the mentally retarded to develop a highly selected group of patients for metabolic 
studies. In 1944, Bernheim and Bernheim demonstrated that the main metabolic path- 
way of phenylalanine was through the parahydroxylation to tyrosine. This opened the 
way for identification of the position of the metabolic error in phenylketonuria. In 
1952, Udenfried and Cooper described the enzymatic system converting phenylalanine 
to tyrosine. The enzyme system was highly specific. D-phenylalanine and many 
derivatives and homologues of 1-phenylalanine were unable to substitute for 1-phenyla- 
lanine as a substrate in this oxidation. In the following year (1953), Jervis demon- 
strated that the specific enzymatic system described by Udenfriend and Cooper was 
absent in phenylketonuria. It was hypothesized that this absence was apparently the 
essential metabolic characteristic of the disease. Udenfriend and Bessman in 1953, 
using loading experiments, showed that phenylketonurics convert only 1/15 as much 
phenylalanine to tyrosine as normal patients. These key experiments by Bernheim, 
Udenfriend, and Jervis initiated subsequent research directed at studying the biochem- 
istry of the enzymatic reaction. While these studies were making significant advances, 
investigations relating the disorder of phenylalanine metabolism to mental retardation 
were slowly being reported. In 1950, ALvord, et al. , described pathological lesions 
of myelin in the brain of phenylketonurics. In 1959, Poser and Bogaert described 
widespread deficient myelination in phenylketonuria. These studies were substantiated 
by Chrome, et al. , in I960. 

Another first occurred in 1951 when Cowie described the first case of "atypical" 
phenylketonuria. The existence of variants of phenylketonuria is still a problem of pri- 
mary importance today. 

Biochemical studies by Mitoma in 1956 began to elucidate the complexity of the 
enzyme system required to convert phenylalanine to tyrosine. Mitoma found the sys- 
tem to consist of two protein components, requiring NAD. Wallace, et al. , in 1957 
confirmed Mitoma's findings. An additional cofactor required to hydroxylate phenyla- 
lanine was identified by Kaufman in 1957. This cofactor, identified as a pteridine, 
was apparently found in normal amounts in phenylketonuria (Kaufman, 1958). In 1961, 
Kaufman identified a pteridine intermediate in the phenylalanine- tyrosine conversion. 
This intermediate was 5, 6-dihydropteridine. The advancement of knowledge related 
to the biochemistry of phenylalanine metabolism, i.e., conversion to tyrosine, led in 
1969 to the development of a reliable, positive assay for the determination of liver 
phenylalanine hydroxylase by Kaufman. A similar assay was developed by Bulitz in 
1969 to determine liver phenylalanine hydroxylase. These assays have been very 
important to the development of differential diagnostic techniques in the detection and 
screening of phenylketonuria. Kaufman and co-workers (Friedman, et al. , 1972) 
subsequently demonstrated directly for the first time that phenylalanine hydroxylase 
was absent in the liver in classical phenylketonuria. One year earlier, Friedman and 
Kaufnnan (1971) had directly shown that phenylalanine hydroxylase activity in normal 
human newborns was as high as in normal adults. Biochemical research of phenylke- 
tonuria has continued with most recent reports (Kaufman, et al. , 1975, and Butler, 
et al. , 1975) demonstrating a phenylketonuria due to a deficiency of dihydropteridine 
reductase. 



0-5 

While investigations at the molecular level on the enzymatic defect have been 
comprehensive, clarification of the nature and pathogenesis of brain damage has been 
somewhat limited. An important contribution in studies of the pathogenesis of brain 
damage was the development of experimental phenylketonuria by Waismen and co- 
workers (Auerbach, et al. , 1958), whose phenylalanine -fed rats showed some retarda- 
tion of learning ability. Experimental phenylketonuria in laboratory animals load-fed 
phenylalanine has been used by Miller et al. (1973) to demonstrate a decrease in pyruvic 
kinase in the brain of the experimental animals. Patel et al. (1973) also suggested de- 
fects in pyruvic metabolism in brain. However, Karrer and Cahilly (1965) convincingly 
demonstrated that studies performed with experimentally induced phenylketonurics 
from forced feeding were inadequate and incorrectly conducted. This caused a halt in 
experimental PKU until the I970's. In 1967, Udenfriend and co-workers (Lipton, et al. ) 
produced a more realistic phenylketonuria model that could be obtained by inhibiting 
phenylalanine hydroxylase with p-chlorophenylalanine followed by phenylalanine loading. 
This significant contribution has led to subsequent studies by Butcher (1970) and others 
who have shown that this model simulates both behavorial and biochemical manifesta- 
tions of phenylketonuria. These experimental models will continue to be used to 
facilitate studies to examine brain defects in phenylketonuria cases. 



Detection and Screening 

Foiling in 1934 was the first to detect and identify urinary phenylpyruvLc acid 
using ferric chloride. It has been said that few diseases are so uniformly and pre- 
cisely identifiable as phenylketonuria. Detection methods have been based primarily 
on either evaluating phenylpyruvic acid concentrations in urine or on determining 
phenylalanine concentrations in serum. Foiling et al. , in 1938, were probably the 
first to determine blood phenylalanine levels using bacterial conversion of phenylalanine 
to phenylpyruvic acid. Most detection methodologies are based on qualitative and/or 
quantitative determination of urinary phenylpyruvic acid or serum phenylalanine. As 
is discussed later, neither of these detection techniques alone gives a sufficiently com- 
prehensive diagnosis of phenylketonuria. The development of paper chromatography 
is perhaps the most important advance associated with detection of metabolic disorders. 
Berry in 1951 used paper chromatography to study metabolic disorders and in 1957 to 
determine phenylalanine. The direct quantitative method of determining serum phenyl- 
alanine became of significant importance following Armstrong's studies in 1956. 
Armstrong and Binkley showed that phenylpyruvic acid in infants could be negative up 
to nearly 1 month. Phenylalanine serum levels increased much earlier. They in fact 
showed that phenylketonuric infants were normal at birth. This has been substantiated 
by many later studies. This fact indicated the need for (1) early screening of infants 
and (2.) a sensitive method of determining serum phenylalanine. It was at this point 
that both improvements in detection methodology and preliminary screening progran"is 
were initiated. Centerwall in 1957 proposed a diaper test for routine screening of 
phenylpyruvic acid in infants. In 1958, Berry et al. initiated a screening program to 
detect phenylpyruvic acid using a filter paper test, and one year later (1959) Gibbs and 
Woolf initiated large-scale screening of urinary phenylpyruvic acid in all infants over 
1 month of age. Prior to these events, urinary screening was limited to patients in 
mental institutions. Even with these limited screening programs, until 196Z the only 
instances in which phenylketonuria was recognized early in infancy occurred when a 
sibling was born in a family with an older retarded child with phenylketonuria. How- 
ever, in 1961, Guthrie first published and introduced a bacterial inhibition assay to 
screen blood for phenylalanine. In 1963, Guthrie and Susi reported on the use of the 



0-6 

bacterial inhibition assay for Large-scale screening. Guthrie's assay for rapid deter- 
mination of phenylalanine in large samples resulted in massive screening programs. 
In 1964, MacCready and Hussey reported on the first statewide screening program 
initiated in 1962 in Massachusetts. Introduction of massive screening programs re- 
sulted in the discovery of hyperphenylalanemia distinct from classic phenylketonuria. 
These variants of phenylketonuria contributed to the confusion of diagnosis. In 1965, 
statements from the American Academy of Pediatrics' Committee on Fetus and New- 
borns recommended blood tests for phenylalanine on all newborns. One year later in 
1966, Berry published guidelines for diagnosis of phenylketonuria. 

In the area of detection methodology, several critical developments occurred 
after 1957. Prior to 1957, phenylalanine was usually determined applying the methods 
developed by Udenfriend and Cooper (1953). In I960, LaDu and Michael introduced an 
enzymatic spectrophotometric test to determine phenylalanine in blood, and in 1962, 
McCaman and Robins introduced a fluorometric method to analyze blood phenylalanine. 
LaDu's enzymatic spectrophotometric test was improved in 1963 to measure phenyl- 
alanine and tyrosine in small amounts of whole blood. 

A significant event that should be considered is the development of phenylalanine 
tolerance tests by Hsia, et al. , in 1956 to determine heterozygotes . The ability to 
detect heterozygotes is of obvious practical theoretical importance in management of 
phenylketonuria. 

At this time, phenylketonuria is screened in 45 of the 50 states within the United 
States and also in most of the European countries as well as in a few countries in Asia, 
the Middle East, and the South Pacific. In the United States, the screening facility 
is usually the central public health laboratory of the state, though in a few states, 
notably California, screening is conducted in relatively small private hospitals. While 
screening programs have been successful, they have also been criticized. Some have 
asked whether the cost of screening equals the benefit of the analysis. Scriver, 1974, 
has stated that the first law of screening stresses that effective measures must reduce 
costs. By successful screening, we could indeed avert the monetary costs to society 
of phenylketonuria by eliminating chronic care in institutions for the mentally retarded. 
At one time, PKU patients composed 1 percent of such residents. It has been pleasing 
to find that $ 1 spent in the screening and treatment of PKU is more than balanced by 
savings of $4 in institutional and other care which would otherwise be given to non- 
screened, untreated retarded PKU patients. This cost-benefit analysis considers only 
the real costs, such as specific costs of screening and treatment, preventing the full 
expression of the disease. This cost should be weighed against the specific life costs 
of traditional or new methods of dealing with the disease without screening. Steiner 
and Smith, in a cost-benefit analysis in 1973, concluded that a PKU screening program 
is beneficial not only to the person who has the disease but also to society. With a 
retrospective approach yielding a cost-benefit ratio of 1:1.66, these authors suggested 
that more money should be funded for this type of research before appropriating huge 
sums for many supposedly worthwhile social programs . However, criticisms of 
massive screening programs continue emphasizing that prevention, treatment, and 
rehabilitation of homozygotes will increase the potential of deleterious genetic traits 
increasing as such persons are allowed to survive and reproduce. The frequency of 
such genes in the population would obviously increase. However, this increase in 
incidence would be only 1 percent in five generations. 



0-7 

Treatmeat of Phenylketonuria 



Treatment of phenylketonuria using low-phenylalanine diets was first suggested 
in 1951 by Woolf and VuUiamy. They also suggested the removal of phenylalanine 
from protein hydrolysates . Dent in 1951 was apparently the first to attempt to treat 
phenylketonurics with phenylalanine- restricted diets; however, during this same year, 
Woolf and Armstrong also began studies on the effect of phenylalanine- restricted diets 
on phenylketonuria. The first reported results were published in 1953 by Bickel, et al. 
In this report and a subsequent report in 1954, Bickel indicated that phenylalanine-free 
and phenylalanine-poor diets resulted in definite improvement in the behavior of a 
phenylketonuric 2-year-old girl. He also found that a return to diets containing phenyl- 
alanine resulted in regression in behavior. Woolf, et al. , in 1955 reported that treat- 
ment of phenylketonuria with a diet low in phenylalanine resulted in a marked intellectual 
improvement in one of his patients. Successful improvement in mental ability was not 
indicated by Horner and Streamer in 1956 and Armstrong, et al. , in 1955 and 1957. 
On the basis of Armstrong's earlier work in 1956 indicating that phenylketonuric infants 
were normal at birth and developed phenylketonuria somewhat after, the need for 
early treatment in infancy was indicated. It is obvious that successful treatment of 
phenylketonuria is dependent upon full and comprehensive diagnosis. During the late 
1950's and the 1960's, considerable confusion existed concerning methodology for 
diagnosis of phenylketonuria. Confusion also reigned in treatment of phenylketonuria 
with reports of chronic phenylalanine deficiency from overtreatment leading to impair- 
ment of cognitive and behavioral development and even to death (Hackney, et al. , 1968; 
Hudson, 1971). In 1958, it was reported that phenylalanine- restricted diets may retard 
growth and in fact be more detrimental to the person than the phenylketonuria itself. 
The participants in active screening and treatment programs realized the seriousness 
of these problems and held a conference in 1966 (Berry, 1967) to discuss the status of 
treatment of phenylketonuria with low-phenylalanine diets. Two major problems were 
repeatedly emphasized: (1) the uncertainty of diagnosis and (2) the need for evaluation 
of dietary therapy. Recommendations were presented to challenge these problems. 

The importance of Snyderman's observations in 1955 on the phenylalanine require- 
ments of infants became of utmost importance. With the introduction of a commercially 
available phenylalanine- restricted formulation called Lofenalac by Mead-Johnson in 
1958, it was potentially possible to treat all diagnosed phenylketonurics. However, a 
certain number of questions existed concerning the maintenance of proper nutrition 
during dietary treatment and the resolution of problems in dietary management of phenyl- 
alanine- restricted diets. The nutritional requirements of phenylketonuric patients 
has been a critical problem in successful treatment. Paine and Hsia (1957) were 
probably the first to attempt to examine connprehensively the dietary requirements and 
tolerance of phenylketonuric patients. Studies related to proper nutrition and growth 
during treatment were subsequently performed by Berry and co-workers. In 1965, 
Umbarger, et al. , developed a list of criteria for successful management of phenyl- 
ketonuria. Still questions existed, and in 1967, Birch and Tizard and Berman, et al. , 
questioned the effectiveness of dietary treatment of phenylketonuria. These many 
questions and others prompted initiation of the National Collaborative Study for Treat- 
ment of Phenylketonuria which began in 1967. Results from this study are still being 
collected. At this time, the need to treat children with phenylketonuria is no longer 
questioned. The much more difficult decision is whether children with hyperphenyl- 
alanemia should have a restricted phenylalanine intake. The success of screening 
and dietary treatment was observed by Spencer in 1974 and MacCready in 1974. 



0-8 

Spencer, in England, noted that no new patients with phenyketonuria had been admitted 
to a large hospital in England for severely mentally handicapped persons since 1968. 
MacCready noted that since screening programs had been initiated in Massachusetts, no 
phenylketonuric children had been admitted to institutions for severely retarded in that 
state. 

Questions, of course, still exist especially with respect to termination of treat- 
ment, maintenance of adequate nutrition for normal physical growth, etc. 



Contributory Events 



Perhaps the most significant contributory event was the introduction of paper 
chromatography by Consden, et al. , in 1944. In 1946, Dent simplified paper chroma- 
tography for analysis of amino acids, and 5 years later (1951), Berry applied paper 
chromatography to the study of metabolic disorders. These developments drastically 
improved the ability to diagnose phenylketonuria and led directly to the identification 
of many inborn errors of metabolism. 

Prior to the development and application of paper chromatography, assays for 
phenylalanine were based on microbial conversion to a derivative (Foiling and Closs, 
1938) which was subsequently determined chemically. These procedures were very 
difficult to apply in clinical laboratories. 

In the area of treatment, the process of Schramm and Primosigh for quantitatively 
removing phenylalanine from protein hydrolysates in 1943 is of significance. This 
process, utilizing charcoal column chromatography, had its inception in 1919 byAbder- 
halden and Fodor, who showed that charcoal could be used to study the adsorption of 
amino acids and peptides. Limited quantities of phenylalanine-free protein hydrolysates 
were made available in 1954. However, commercial processes to enzymatically 
remove phenylalanine from protein hydrolysates came into existence in the United States 
in 1958. 

An event that may be considered contributory and of great importance was the 
development of the dried blood spot by Guthrie in 1961. Although initially developed 
for use in Guthrie's inhibition assay for phenylalanine, the dried blood spot has gained 
wide usage in essentially all massive screening programs for PKU, providing a simple, 
convenient method for obtaining blood samples for analysis. 

Another contributory event in the area of dietary treatment was Snyderman's 
report on the phenylalanine requirements of infants in 1955. Results from this study 
provided information leading to the development of nutritionally balanced diets to treat 
phenylketonuria in infants. Snyderman's work followed years of investigations by many 
workers, primarily Rose, to identify essential amino acids (the last, threonine, 
identified in 1937) and to determine their requirements in man. 



ADDENDUM 



Since this text was drafted, a very important publication from the National Acad- 
emy of Sciences entitled Genetic Screening: Programs, Principles, and Research 



0-9 

has been obtained. The report prepared by the Committee for the Study of Inborn 
Errors and Metabolism reviews investigations into the origin, history, and current 
standing of screening for PKU and into the effectiveness of its treatment. The report 
aLso covers educational, legislative, and organizational aspects of genetic screening 
and provides an excellent reference for the current status of PKU research. 



REFERENCES 



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Alvord, E. C, Jr., Stevenson, L. D., Vogel, F. S., and Eagle, R. L. , Jr., J. 
Neuropath. Exper. Neurol., 9, 298(1950). 

American Academy of Pediatrics, Pediatrics, 35, 499 (1965). 

Armstrong, M. D. , and Binkley, E. J., Jr., Proc. Soc. Exp. Biol. Med., _93, 418 
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Armstrong, M. D. , Low, N. L. , and Bosma, J. F. , Amer. J. Clin. Nutri. , 5, 543 
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Armstrong, M. D., and Tyler, F. H. , J. Clin. Invest., 34, 565(1955). 

Auerbach, V. H. , Waisman, H. H. , and Wyckoff, L. B., Nature, J_82, 871 (1958). 

Bernheim, M.L.C., and Bernheim, F. , J. Biol. Chem. , 152 , 481 (1944). 

Berry, H. K. , Sutton, H. E., Cain, L. , and Berry, J. S., Univ. Texas Publication 
No. 5109, 22 (1951). 

Berry, H. K., Proc. Soc. Exp. Biol. Med., 95, "71 (1957). 

Berry, H. K. , Sutherland, B., Guest, G. M., and Warkany, J., JAMA, 167 , 2189 
(1958). 

Berry, H. K. , Umbarger, R., and Sutherland, B. S., Pediatrics, 2j_(6), 929(1958). 

Berry, H. K. , and Wright, S., J. Pediatrics, ^0, 142 (1967). 

Bickel, H., Gerrard, J., andNickmans, E. M. , Lancet, 2, 812(1953). 

Bulitz, C, Biochim. Biophys. Acta, 191 , 249 (1969). 

Butcher, R. E., Nature, _226, 555 (1970). 

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Cowie, V. A., Lancet, 2, 272 (1951). 

Dent, C. E., Lancet, 2, 637 (1946). 



O-IO 

Dodge, P. R., Mancall, E. L,. , Crawford, J. D. , Knapp, J., aad Paine, R. S., New 
Eng, J. Med., 260, 1104(1959). 

Efron, H. L. , Young, D. , Moser, H. W., and MacCready, R. H. , New Eng. J. Med., 
270 , 1378 (1964). 

Foiling, A., Ztschr. f. Physiol. Chem. , 227 , 169(1934). 

Foiling, A., and Closs, K., Hoppe Seylers Z . Physiol. Chem., 254 , 115.(1938). 

Foiling, A., Closs, K. , and Gammnes, T. , Hoppe Seylers Z. Physiol. Chem., 256, 
1 (1938). 

Friedman, P. A., and Kaufman, S., Arch. Biochem. Biophys., 146 , 321 (1971). 

Friedman, P. A., Kaufman, S., and Kang, E., Nature, 240 , 157 (1972). 

Garrod, A. E., Lancet, 2, 1-7, 73-9, 142-8, 214-20(1908). 

Gibbs, N. K., and Woolf, L. I., Brit. Med. J., 2, 532(1959). 

Guthrie, R. , JAMA, 17_8, 863(1961). 

Guthrie, R. , and Susi, A., Pediatrics, 32, 338(1963). 

Hackney, 1. M. , Hanley, W. R. , Davidson, W., and Linsao, L. , J. Pediat. , 72, 646 
(1968). ~ 

Horner, F. A., and Streamer, C. W., JAMA, j_61, 1628(1956). 

Horner, F. A., Streamer, C. W., Alejandrino, L. L. , Reed, L. H. , and Ibbott, F. , 
New Eng. J. Med., _266, 79 (1962). 

Hsia, D.Y-Y., Develop. Med. Child. Neur., 9, 531 (1967). 

Hsia, D.Y-Y., Progress in Medical Genetics, II, Steinberg, A. G. , and Bean, A. G., 
editors, Grune & Stratton, New York (1970). ~ 

Hsia, D.Y-Y., and Dobson, J., Lancet, J., 905(1970). 

Hsia, D.Y-Y., DriscoU, K. W., Troll, W., and Knox, W. E., Nature (London), 17 8 , 
1239 (1956), 

Hudson, F. P., in Phenylketonuria and Other Inborn Errors of Amino Acid Metabolism , 
Bickel, H. , Hudson, F, P., and Woolf, L. I., editors, Geor. Thieme Verlag, 
Stuttgart (1971). 

Hudson, F. P., Mordaunt, V. L. , and Leahy, I., Arch. Dis. Child., ^(239), 5 
(1970). 

Jervis, G. A., J. Ment. Sci. , 85, 719 (1939). 

Jervis, G. A., J. Biol. Chem., _1_69, 651 (1947). 

Jervis, G. A., Proc. Soc. Exp. Biol. Med., 1_S, 83 (1950). 

Jervis, G. A., Proc. Soc. Exp. Biol. Med., _82, 514(1953). 

Jervis, G. A., in Genetics and Inheritance of Integrated Neurological and Psychiatric 
Patterns , Hooker, D., and Hare, C. C, editors. Res. Nerv. Ment. Dis., 33, 259 
(1954). ~ 

Justice, P., O'Flynn, M. E., and Hsia, D.Y-Y., Lancet, _1, 928(1967). 

Kapellar-Adler, R. , Biochem. Z., 252, 185(1932). 



O-ll 

Karrer, R. , andCahiLLy, G. , Psych. BuLletin, _64, 52 (1955). 

Kaufman, S., J. Biol. Chem. , 230 , 931 (1958). 

Kaufman, S., J. Biol. Chem. , 236, 804(1961). 

Kaufman, S., Arch. Biochem. , J_34, 219(1969). 

Kaufman, S. , HoLtzman, N. A., Milstien, S. , Butler, I. J., and Krumholz, A., New 
Eng. J. Med., 293 (16), 786 (1975). 

LaDu, B. N., and Michael, P. J., J. Lab. Clin. Med., SS_, 491 (I960). 

Levy, H. L, , Karolkewicz, V., Houghton, S. A., and MacCready, R. A., New Eng. 
J. Med., _282, 1455 (1970). 

Lipton, M. A., Gordon, R. , Gvroff, G., and Udenfriend, S, , Science, 1 56 , 248(1967). 

MacCready, R. A., andHussey, M. G. , Am. J. Pub. Health, 54, 2075 (1964). 

McCaman, M. W. , and Robins, E., J. Lab. Clin. Med., ^, 885(1962). 

Miller, A. L. , Hawkins, R. A., and Veech, A. L. , Science, j_79, 904(1973). 

Mitoma, C, Arch. Biochim. Biophys., _60, 476 (1956). 

Mitoma, C, Auld, R. M., and Udenfriend, S., Proc. Soc. Exp. Biol. Med., ^, 634 
(1957). 

Paine, R. S., andHsia, D.Y-Y., Am. J. Dis. Child., _94, 224(1957). 

Patel, M. S., Grover, W. D., and Averback, V. H., J. Neurochem. , 20, 289 (1973). 

Penrose, L. S. , Lancet, J., 23 (1935). 

Penrose, L. S., and Quastel, J. H. , Biochem. J., 3j., 266(1937). 

Poser, C. M., andBogaert, L. van. Brain, _82, I (1959). 

Schramm, G. , and Primosigh, J., Ber. Deutsch. Chem. Ges., 76, 373 (1943). 

Scriver, C. R. , Pediatrics, _54, 616 (1974). 

Snyderman, S. E., Pratt, E. L. , Cheung, M. W. , Norton, P., Holt, L. , Hansen, 
A. E., and Painos, T. C, J. Nutrition, bb_, 253 (1955). 

Spencer, D. H, , Lancet, j., 1351 (1974). 

Steiner, K. C, Smith, H. H. , Inquiry, 2^, 34(1973). 

Umbarger, B., Berry, H. K. , and Sutherland, B. S., JAMA, r93, 784(1965). 

Udenfriend, S. , and Cooper, J. R. , J. Biol. Chem., 194 , 503 (1952). 

Udenfriend, S., and Cooper, J. R., J. Biol. Chem., 203, 953 (1953). 

Wallace, Z. W., Moldave, K. , and Meister, A., Proc. Soc. Exp. Biol. Med., 94, 
632 (1957). 

Woolf, L. 1., Griffiths, R., and Moncrieff, A. A., Brit. Med. J., j., 57 (1955). 

Woolf, L. I., Griffiths, R., Moncrieff, A. A., Coats, S. , and Dillistone, F., Arch. 
Dis. Child. , _33, 31 (1958). 

Woolf, L. I., and Vulliamy, D. G. , Arch. Dis. Child., 26, 487(1951). 



h 



DISCOVI 



1908 Gorrod 



DISCOVERY AND ETIOLOGY 



DIAGNOSIS AMD SCREENING 



. 1934 FBllIng DriD 



- 1335 Pffnoio Conll 



MR/S/W/G,P/M 



. 1938 FBIIrna PMnylo 



- 1937 PinioH Osnl 



-1938 faillna Moalur. 



NR/S/U/G/GL 
19&2 UiUrtlirind DMcriMi anivma iviicm au- 

NR/D/U/G/GL 

MR/D/U/G/GL 

1953 Uctarrriind Loadina cipEiirninii ihow PKU 

aetiv.ly in PKU 

MR/S/U/G/GL 



I DV/SyW/P/1 

1*16 Ooni SimpliUM MIK' thromaIEBirj[ 



- 1951 Berry Appllci lupur dirsinaiograjil 
DV/D/U/G/M 



MR/S/U/G/GL 



CODES 

NR Nonmlcion-Orienied Rnearch 

MR Mlssion-Oilented Research 

OV Oevelopmenl 

S Signiliiant Event 

Oecitrve Evertl 
U U.S. Evenl 
W Foteign Eveni 
G Governmem Funding 
P Privsle Funding 
M Mridical School - Hoipilal - 

Univetiity 

1 Rneaich Intlitulion 
GL Governmem Laboralory 
C Companv 



TREATMENT 



JO mm QuiniUMinly ramovn phtiiylilmini 
OV/S/W/P/I 



enyiilmirn die II : luggau iti 
pncnUoloni™ from piolein 

MR/D/W/P/M 



Jl PKU. protein hytfro 
MR/O/W/ /M 



ml. «ih(Kji ph<r„Hei 
MR/S/U/P.G/M 



MR/S/U/G/GL 



1970 Buictxr Anirrul rmxMli bond on p<hioraph*n< 

sod bliKhimli^l mini I mail oil o1 FKl 
MR/S/U/G/M 

MR/S/U/G/GL 




PROBLEMS IN 
TREATMENT 

■DENTIFIED 



EFFECTIVENESS 
OF TREATMENT 
QUESTIONED 



SUCCESS 
DOCUMENTED 



HISTORIOGRAPH -PHENYLKETONURIA 



180' 



p-1 

APPENDIX P. AUSTRALIA ANTIGEN 
INTRODUCTION AND SUMMARY 



In 1972, it ■was estimated that transfused blood -was responsible for more than 
3 0, 000 cases of overt hepatitis and as many as 3, 000 deaths per year in the United 
States (Marston, 1972). The disease had been characterized and linked to the in- 
jection of blood products since the 1930's, yet all attempts to solve the problem 
through the usual routes of developing animal models or propagating the responsible 
agent in tissue culture had failed. The problem of identifying asymptomatic infective 
donors appeared insoluble. Then a medical geneticist working in an unrelated area 
of investigation made a key observation. The link between viral hepatitis and a new 
antigen which had been discovered in the serum of an Australian aborigine was re- 
ported in 1967 and quickly confirmed. New life was breathed into the lagging search 
for a ■way to control this important disease. 

With the backing of the National Institutes of Health, the development of this 
discovery moved quickly from the bench to practical application. By 1972, all 
federally licensed blood banks -were required to test for the presence of Australia 
antigen. While advances in knowledge about the disease and its causative agent 
continue to be made and refinements in testing methods implemented, the develop- 
ment of this discovery is already saving lives by preventing the use of infective 
blood that less than 5 years ago ■would not have been detected. Posttransfusion 
hepatitis has virtually been eliminated. 



HISTORICAL ACCOUNT 



Introduction 



While viral hepatitis has an ancient history under numerous aliases such as 
"infectious hepatitis", "epidemic jaundice", and "acute catarrhal jaundice", the 
first outbreak which can be associated with the injection of a human blood product 
■was reported by Lurman in 1885. This reported outbreak followed the use of 
glycerinated huraan lymph in a smallpox vaccine. It ■was not until the late 1930's 
and early 1940's that evidence began to be accumulated which brought this assoc- 
iation to light. In the years that followed, a multiplicity of terms appeared ■which 
were used to describe the disease associated with the use of blood and blood 
products. Some of the most common terms ■were "serum hepatitis", "homologous 
serum jaundice", "long incubation hepatitis" , and "posttransfusion hepatitis". 

We no^w recognize that there are at least •two distinct viral hepatitis diseases. 
The first, believed to be caused by hepatitis A virus, is termed viral hepatitis, 
T^ype A. The second, ■which is caused by hepatitis B virus, is no^w termed viral 
hepatitis, T^ype B. While the routes of transmission are now recognized to 
overlap for the two agents, in general, viral hepatitis, Type A is s^ynonymous 
with "infectious hepatitis", while Type B is what has usually been called "serum 
hepatitis". 



P-2 

This report is concerned primarily with viral hepatitis, Type B because one of 
the morphologic formis of the Australia antigen is in fact believed to be the hepatitis 
B virus. In keeping with current terminology, hepatitis B surface antigen (HBgAg) 
will be used interchangeably with the terms Australia antigen and hepatitis B virus. 

While research on HBgAg continues at a rapid pace today, not only in terms of its 
specific role in viral hepatitis but also in terms of its potential involvement in other 
diseases of the liver, the scope of this account is limited to those events that led to the 
widespread use of tests for HBgAg for screening blood to be used for transfusion and in 
the preparation of biological products. 

Development of Viral Etiology 



The reports of MacNalty in 1937 and Propert in 1938 are representative of those 
which first linked hepatitis in humans with the use of human serum in injectables. In 
both cases, it was reported that hepatitis followed inoculation of children with con- 
valescent measles serum containing human serum. These reports were shortly fol- 
lowed by similar accounts of hepatitis associated with the use of yellow fever vaccine 
containing human serum (Findlay, 1938; Sopor, 1938; Fox, 1942). These workers 
recognized both the similarities and the differences of the etiology of this disease with 
"infectious hepatitis". By 1942 (anon, editorial), it was recognized that 28, 585 cases 
of hepatitis in United States' military personnel could be traced to the use of human 
serum in yellow fever vaccine. Canneron (1943) was the first to demonstrate that the 
inoculation of human volunteers with serum from hepatitis patients resulted in the 
transmission of the disease. His studies were carried out in a British field hospital 
in Palestine, but the results were compromised by the difficulty in controlling the ex- 
periment and following up on the subjects. The same year, Oliphant (1943) confirmed 
these findings in a controlled study in the United States. He demonstrated by trans- 
mission experimients in human volunteers that the causative agent was in the donor 
blood before jaundice appeared. Oliphant also attempted to infect monkeys, pigs, 
rabbits, guinea pigs, rats, mice, and hamsters without success. This was the first 
of many unsuccessful attempts to develop an animal model for the disease. The fol- 
lowing year, Neefe (1944) reported on the 100 percent incidence of hepatitis in nine 
volunteers inoculated with human plasma and serum associated with hepatitis outbreaks. 

The results of these studies proi-npted other workers to examine the similarities 
and differences between this new "serum hepatitis" and the disease "infectious hepa- 
titis". In 1945, Havens reported on a lack of cross immunity between "infectious 
hepatitis " and "serum hepatitis". The same year, Paul noted that there appeared to 
be a consistent difference in the incubation period between the two forms of hepatitis. 
While transinission by oral ingestion of feces was confirmed for "infectious hepatitis", 
this did not appear to be the case for "serum hepatitis" (Neefe, 1945a). Extending 
Haven's work, Neefe (1945b) confirmed the lack of cross immunity between the two 
diseases while demonstrating homologous immunity for both "serum" and "infectious" 
hepatitis. On the basis of the etiology of disease in exposed volunteers, Neefe (1946) 
proposed tliat two different viral agents were responsible for "serum hepatitis" and 
"infectious hepatitis". Francis (1946) confirmed Olipliant's finding that hepatitis could 



P-3 

be transferred by transfusion when the donor was still in the pre symptomatic period of 
hepatitis. The asymptomatic carrier state of hepatitis was demonstrated by human 
volunteer studies by Murray in 1954. 

During this period of time, no one was successful either in developing an 
animal model for the disease or in cultivating the viral agent in tissue culture. 
The first successful transmission experiments in animals were not reported until 
1970 (London, 1970). This was a severe limitation on studies to define the nature 
of the responsible agent. Studies on the etiology of the disease in human volunteers 
continued for the next 15 years. While a great deal of infornnation -was generated 
by these studies, much confusion persisted, and this line of research did not result 
in the discovery of the causative agent. Indeed, the discovery was precipitated 
by an entirely unrelated line of investigation. 



Polymorphism 



As early as 1902, Schiitze had demonstrated the formation of isoantibodies 
in transfused rabbits. In 1916, Marie reported on two patients who formed anti- 
bodies against certain human sera when injected intravenously. In 1929, Gyorgy 
used complement- fixation techniques to demonstrate that a miultiply transfused 
patient developed antibodies against the serum of one of the donors. However, 
it was not until the mid-1950's that genetically controlled variations in blood- serum 
proteins began to be identified. Oudin (1956) dem.onstrated in rabbits and Grubb (1957) 
in humans the variations of -y- globulin types, while Sm^ithies (1959) described the 
genetically controlled variations in haptoglobins and transferrins. The same year, 
Hirschfeld published on group specific precipitating components in the a2 region of 
human sera. 

At the National Institute of Arthritis and Metabolic Diseases, Dr. Blumberg 
found these reports of great interest. He was personally interested in genetic 
variations. While Dr. Allison of the National Institute for Medical Research, 
London, was a visiting scientist at NIAMD, he and Dr. Blumberg collaborated on 
a study to determine whether the genetic variations in serum proteins in man were 
sufficiently different to induce isoimmunization upon transfusion and to form pre- 
cipitating antibodies as had been demonstrated in rabbits by Oudin (1956). Blumberg 
had assembled a collection of sera from many different population groups in many 
parts of the world at the Geographic Medicine and Genetics Section of the NIAMD, 
and panels of 24 of these sera were tested against the sera of multiply transfused 
patients at the NIH Clinical Center. They were in fact able to demonstrate an 
antibody reaction to several of the test-panel sera when tested against one of 
the patient's serum (Allison, 1961). In 1962, Blumberg demonstrated the exist- 
ence of a genetic polymorphism in human low-density /3- lipoprotein. This quickly 
led to the discovery of a second antiserum to low-density /3-lipoprotein (Blumberg, 
1963) and thus began a course of investigation which was to lead to the discovery of 
the Australia antigen. 



P-4 
Discovery 

In 1964, Blumberg first disclosed that an isoprecipitin had been discovered in 
the serum of multiply transfused hemophilia patients that reacted with a rare protein 
in the sera of Australian aborigines and leukemia patients. In 1965, Blumberg, who 
had begun working at the Institute for Cancer Research (ICR) in Philadelphia the pre- 
vious year, published again on this new antigen which had now been named Australia 
antigen. He described the absence of Australia antigen in normal U. S. subjects and 
the high frequency in some foreign populations and in acute leukemia patients. By 
1966, Blum.berg and his co-workers had evidence that the Australia antigen was 
different from typical low- or high-density lipoproteins (Alter, 1966). 

A series of studies was initiated to determine the distribution of this new antigen. 
Family clustering was found that suggested simple autosom.al inheritance but was also 
consistent with an infective agent (Blumberg, 1966). The high frequency of occurrence 
of the antigen in sera of acute leukemia patients led to speculation that individuals 
with the Australia antigen trait may be more susceptible to leukemia than those without 
it. It was reasoned, therefore, that individuals with a high risk of developing leukemia, 
such as patients with Down's syndrome, raight constitute a population having a higher 
than normal frequency of the antigen. In testing this hypothesis, Blumberg (1967) 
did, indeed, find a high frequency of the Australia antigen in Down's syndrome patients. 
However, he and his colleagues also discovered that the antigen was associated -with 
viral hepatitis. 

The initial discovery of the possible relationship between HBgAg and hepatitis 
■was made by chance. A 15-year-old Down's syndrome patient who had tested negative 
for HBgAg in 1965 was retested in 1966 and found to be positive. This observation 
intrigued the investigators and they arranged for the patient to be admitted to the 
Clinical Research Unit of the Institute for Cancer Research for further study. Since 
HBgAs was thought to be a serum protein at that time, they evaluated the liver func- 
tion in the patient and discovered an elevated serum glutamic pyruvic transaminase 
(SOFT). Following through with a liver biopsy, they confirmed hepatitis (London, 1972). 
This finding led to the hypothesis that HBgAg is associated with viral hepatitis. Other 
Down's syndrome patients with and without HB Ag were checked for the disease, and 
indeed, there was a higher incidence of HBgAg in those patients with hepatitis (Blumberg, 
1967). 



Confirmation and Significance 



Blumberg 's observation breathed new life into the investigation of this disease, 
since now there was a serological marker of infection. Expanded studies at ICR con- 
firmed the high incidence of HB_Ag in patients with Down's syadrome and associated 
elevated SGPT levels (Sutnick, 1968). In Japan, Okochi (1968) reported on the fre- 
quency of HBgAg in Japanese blood donors. He also documented the appearance and 
subsequent disappearance of the antigen in transfused patients. Prince (1968a) re- 
ported on the presence of what he called SH antigen in the serum of patients during the 
incubation period of posttransfusion hepatitis. The same year it was reported that this 
antigen was in fact the same as the Australian antigen described by Blumberg (Prince, 



P-5 

1968b). Also in 1968, Bayer reported on the discovery of virus-like particles I90 to 
210 A in diameter in HBgAg-positive serum. This immediately gave support to the 
theory that the antigen was in fact the virus responsible for the disease. 

By 1969, four major lines of research activity had developed along with a number 
of minor thrusts. One major line of investigation was the continued study of the asso- 
ciation of the antigen with hepatitis in transfused patients and the potential for using 
HBgAg as an indicator to identify infected blood donors. Closely associated with this 
effort was the continuing effort to develop analytical techniques of improved sensitivity 
and specificity. These two lines of work eventually meshed and led to the use of HB Ag 
testing by all American Red Cross and federally licensed blood banks. 

The third line of researcb activity, which was supportive in nature, was the 
further study of the nature of the antigen and its structural properties which grew out 
of Bayer's report. The fourth major line of investigation, -which -was really an exten- 
sion of the epidemiology experiments of the 1940's and 1950's combined with the new 
knowledge of the role of HBgAg, was the continued study of the differences between 
"serum." and "infectious" hepatitis. 

One side issue which continued to be followed up by Blumberg's group was the 
hypothesis that HBgAg was an autosomal recessive trait. This has been represented 
on the historiograph because it rounds out the original work on leukemia patients which 
led to the initial discovery. Other areas of research which have not been included on 
the historiograph include the attempts to develop animal models and tissue-culture 
systems. "While some progress has been made in both of these areas, the work was 
not contributory to the development of the HBgAg test for viral hepatitis, Type B. 
Another area of activity which is still being pursued, and which may have important 
practical applications in the future, is the study of hepatitis viral subtypes. This 
area is not included in this report because it did not have a bearing on the practical 
application of the HBgAg test. 



Physical Properties 



Wbile a great deal of work has been done to characterize the physical properties 
of the HB Ag, only a few key representative papers have been cited on the historiograph. 
These are sufficient to put into perspective the base of knowledge that was building in 
1969 and 1970 which gave credence to the antigen as a viral-like agent. It is interest- 
ing to note that the negative staining method for high-resolution electron microscopy 
of viruses, which was applied in these studies, was developed in 1959 by Brenner in 
England and would not have been available to earlier workers. 

Following Bayer's report in 1968, Hirshman (I969) documented the high fre- 
quency of HBgAg during the acute phase of "serum hepatitis", and confirmed the virus - 
like structure of the associated particles by electron microscopy. These two reports 
were followed by four other significant studies, all of which were reported the follow- 
ing year. Perhaps the most significant was the discovery by Dane (1970) of 42-nm 
virus -like particles in HBgAg-positive serum from hepatitis patients. He was the first 
to suggest that these larger particles might be the complete virus, rather than the 
smaller particles noted by Bayer and Hirshman which could be excess outer layer 



P-6 

material sloughed from the larger particles. Jokelainen (1970) confirmed Dane's find- 
ings and presented evidence that the core of the large particles contained nucleic acid, 
while the small particles contained only membrane material. Almeida (1970) first re- 
ported the presence of virus-like particles in the livers of HB Ag-positive patients, 
and Coyne (1970) demonstrated the association between the presence of fluorescent 
particles in the liver cells and presence of HBgAg in serum. In 1970, Almeida reported 
on distinct antibody reactions to the core of the Dane particle and outer coat. 

As stated earlier, for the purposes of this report, these initial studies of the 
physical properties of the antigen serve to illustrate the nature of the work which identi- 
fied the virus -like structure of the HBgAg and gave further support to the hypothesis 
that the antigen was the causative agent of viral hepatitis. Type B. For further insight 
into the current understanding of the complex nature of the antigen, the reader is re- 
ferred to the following pertinent papers: LeBouvier, 1971; LeBouvier, 1973; Hoofnagle, 
1973; Kaplan, 1973; Zuckerman, 1974; Mazzur, 1974; Gerety, 1975; and Magnius, 1975. 



Two-Virus Theory 



In a previous section of this appendix we reviewed the early epidemiological 
studies which demonstrated the direct connection between the use of human serum in 
injectables and the contracting of viral hepatitis. It was pointed out that while much 
was learned about the etiology of the disease, the human-volunteer studies did not lead 
to the solution of the problem. It is interesting to note that studies of this type were 
still being reported in 1967, the same year that Blumberg demonstrated the link be- 
tween HBsAg and hepatitis. Prior to this discovery, relatively little progress had been 
made since the 1940's. For example Krugman's report in 1967 that "serum hepatitis" 
was infective by mouth, as opposed to Neefe's (1945a) earlier findings, only added to 
the general state of confusion. The 1967 report citing evidence for two distinct types 
of infectious hepatitis having different incubation periods simply 'confirmed Paul's find- 
ings of 1945. Following the discovery of the HBgAg, however, things began to fall into 
place. In 1969, Giles reported that HBgAg was present in pooled serum from "serum 
hepatitis" (MS-2) patients but not in "infectious hepatitis" (MS-1) patients. Krugman 
(1970) detected HBgAg in 39 out of 40 patients with viral hepatitis. Type B (MS-2), but 
in none of 41 patients with viral hepatitis. Type A (MS-1). He also demonstrated that 
y-globulin neutralized infectivity of Type B serum but not of Type A. Prince (1970a) 
and Lander (1971a) confirmed the fact that HBgAg was present in Type B but not in 
Type A viral hepatitis. These and other studies quickly established that there were 
basically two forms of viral hepatitis and that viral hepatitis Type B was the type in- 
volved with the use of blood and blood products, and associated with the HBgAg. 



Development and Application of HBgAg Tests 



During I969 and 1970, several studies were reported which convincingly tied 
the HBgAg to hepatitis following blood transfusion. Gocke (1969) reported a close 
correlation of titre of antigen with the course of hepatitis and documented hepatitis 
in three of eight patients who received HBgAg-positive blood. Nordenfelt (1969) 



P-7 

reported the presence of HBgAg in 70 percent of hepatitis patients tested and noted the 
unusual persistence of antigen in dialysis patients. In 1970, Barker demonstrated the 
association of HBgAg with hepatitis by direct inoculation studies in volunteers. How- 
ever, he also demonstrated that transmission was possible at antigen levels too low to 
be detected by complement fixation. The same year, Okochi (1970a) reported on the 
statistically higher occurrence of posttransfusion hepatitis in recipients of HBgAg- 
positive blood. He, too, stressed the need for more-sensitive detection techniques. 
Thus, while the HBgAg was clearly implicated in the transmission of viral hepatitis, 
Type B, miethods were not available which would guarantee HBgAg-negative blood. On 
this basis, in 1970, a Panel of the Committee on Plasma and Plasma Substitutes of the 
Division of Medical Sciences, National Academy of Sciences — National Research 
Council published a statement (NAS-NRC, 1970) which presented their conclusion that 
nonuniformity of tests for HBgAg precluded its general adoption by blood-banking and 
transfusion services. At this point, therefore, it is germane to review the course of 
development of HBgAg-measuring techniques. 

When Blumberg and Allison began their search for isoprecipitins in human sera, 
they adapted the agar-gel-diffusion technique (AGD) of Ouchterlony (1958). This tech- 
nique had the advantage of being simple to perform, required little equipment, and 
allowed large numbers of samples to be studied as opposed to the alder, one -dimensional 
gel diffusion methods. This method was employed in all of Blumberg's early work 
(Allison, 1961; Blumberg, 1962; Blumberg, 1963; Blumberg, 1964; Blumberg, 1965; 
Alter, 1966; Blumberg, 1966; Blumberg, 1967; Sutnick, 1968; Blumberg, 1969; and 
Sutnick, 1970), by Okochi (1968, 1970a), Prince (1968a, 1968b), Cocke (1969), and 
Nordenfelt (196 9). While the AGD method had the advantage of giving no false positives, 
it was relatively insensitive and required 1 to 3 days to complete. 

In 1969, Shulman reported on the use of complement fixation (CF) to measure 
HBgAg, and Purcell demonstrated that the microtitre CF method as modified by Sever 
(1962) was 24 times as sensitive as AGD. However, the test required a supply of active 
complement and Anti-HBg which was not generally available. Other disadvantages of 
the method include the need to run several dilutions in order to avoid the prozone reac- 
tions with high-titre sera and the potential for anticomplementary activity of some sera 
due to antigen-antibody complexes or other factors (Schmidt, 1970). Also, as shown 
by Barker (1970), the CF method was still not sensitive enough to detect infective levels 
of HBgAg. 

In 1970, several advances were made in methodology for measuring HBgAg. 
Counterimimunoelectrophoresis (CEP) techniques were described by Gocke (1970), 
Pesendorfer (1970), and Prince (1970b), which were approximately 10 times as sensi- 
tive as AGD methods. While still being less sensitive than 'desired, the method was 
easy to use and therefore more attractive than CF techniques for large-scale screening 
of blood products. The same year, Vyas ( 1970) and Okochi (1970b) described hemag- 
glutination methods, based on the earlier work of Nelson (1953), which were more 
sensitive than AGD or CEP methods; and Walsh (1970) published the first report on the 
use of radioim.munoassay (RIA ) to measure HBgAg and Anti-HBg, as adapted from the 
earlier work of Yalow (I960) and Utiger (1962). 

In light of the rapid advances being made in the state of the art for detecting 
HBgAg and the potential public-health significance of screening out infective blood 
products, the NAS-NRC panel modified its position in 1971 and recommended the 
testing of all donor blood for HBgAg while pointing out the limitations imposed by 
the still limited supply of standard antisera (NAS-NRC, 1971a). Also in 1971, the 
Food and Drug Administration (FDA) established the first standards for Anti-HBg. 



P-8 

The same year, the American Red Cross took a stand and initiated testing of all blood 
donors using the CEP method. 

In April, 1971, the NAS-NRC panel published another statement on the use of 
routine testing in blood banks based on the results of a comparative-sensitivity study 
of HBgAg tests involving 20 laboratories (NAS-NRC, 1971b). They recommended that 
all blood banks begin testing using the AGD or CEP method, depending on how quickly 
the results were needed. They also recommended that CF, indirect hemagglutination 
inhibition (HAI), or RIA tests not be adopted unless procedures were developed that 
made them practical. This recomraendation was based on the fact that only a few blood 
banks had the technical capability to perform these tests. It was not until July of the 
following year that the FDA (1972a) required that all federally licensed blood banks test 
for the HBgAg using a method at least as sensitive as AGD. 

This action by the FDA may be considered the culmination of Blunaberg's dis- 
covery of the Australia antigen. However, it is important to review some of the events 
which followed. If the development of the testing methodology had not proceeded past 
the use of AGD and CEP, the procedure would have screened out perhaps only 25 per- 
cent of the HBgAg donors, for these methods were relatively insensitive. 

Following the pioneering work by Walsh (1970) on the application of RIA for 
measuring HBgAg, there was a flurry of activity in several laboratories to improve 
this technique (Lander, 1971b; CoUer, 1971; HoUinger, 1971; Ling, 1972). Abbott 
Laboratories developed a solid-phase RIA test which was 1000 times more sensitive 
than the AGD method (Ling, 1972). In 1972, the FDA (1972b) licensed the first reagent 
for the RIA method which was marketed by Abbott under the name of Ausria. During 
the same period, continual evidence v/as accumulating on the superiority of the CEP 
method over AGD, and the FDA (1972c) ruled that all federally licensed blood banks 
must test for HBgAg using a procedure at least as sensitive as CEP. In 1973, the 
American Red Cross initiated the use of RIA using the Ausria test. Meanwhile, evi- 
dence was miounting that the RIA test was giving false positive results (Prince, 1973; 
Vyas, 1973; Alter, 1973; Irwin, 1974). The American Red Cross responded quickly 
as new information became available by initiating guinea-pig-serum neutralization pro- 
cedures in 1973, and by the use of the bead-RIA test with labeled human antibody in 
1974 to increase specificity and reduce false positives. The problem of false positive 
results is important not only because of the unnecessary loss of blood donors, but 
more importantly because of the unnecessary hardship caused to the donor, including 
the worry and expense of follow-up examinations for a nonexistent carrier state and 
even the potential exclusion from certain job categories. Investigations continue today 
on methods to improve the specificity of the RIA test (Ling, 1975). 

The Bureau of Biologies of the FDA is also still actively monitoring the latest 
improvements in reagents and test procedures, and modifying the regulations as ap- 
propriate. In April, 1974, licensing procedures were established for antibody for use 
in the reverse passive hemagglutination (RPHA) test (FDA, 1974). In July, 1975, the 
FDA ruled that all federally licensed blood banks be required to use test procedures 
equivalent in sensitivity to the RPHA or RIA tests effective September 15, 1975 (FDA, 
1975). 



P-9 
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P-10 

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p-11 

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P-12 



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P-13 

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I 



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POLYMORPHISM 



DISCOVERY AND DETECTION 



PHYSICAL PROPERTIES 



DETECTION METHODS 



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Q-1 

APPENDIX Q. GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY 

INTRODUCTION AND SUMMARY 

A deficiency in red cell enzyme activity of glucose -6-phosphate dehydrogenase 
(G6PD) is a common occurrence throughout the world. It is estimated that over 
100, 000, 000 people are affected by this deficiency. In this country, nearly 3, 000, 000 
Americans have the trait, and the majority of these are blacks. G6PD deficiency is 
probably the most prevalent hereditary enzyme defect of clinical significance. 

The consequences of G6PD deficiency are varied, the most common being a 
hemolytic episode which occurs when the affected individual is exposed to an external 
influence such as a drug, an environmental pollutant, or certain foods. Inheritance of 
the G6PD trait has been shown to be X- linked. 

The incidence of hemolytic episodes has been associated for many years with the 
use of certain drugs or the exposure to environmental agents. In the 5th century B. C. , 
Herodotus warned against the use of fava beans. Sensitivity to naphthalene of people in 
the Mediterranean basin has been noted in more recent times. The understanding of the 
mechanism of G6 PD deficiency has brought together a heterogeneous group of seemingly 
unrelated syndromes and linked them to a common source. From a clinical standpoint, 
the research done on G6PD deficiency enabled the trait to be easily detected in a given 
individual. After detection of the trait, the use of drugs that cause hemolysis in sensi- 
tive patients can be avoided. A therapeutic method for treatnaent of the anemia resulting 
from certain rare types of G6PD deficiency does not exist. 

One of the most important aspects of the research done on G6PD deficiency has 
been its use as a marker in studies of human genetics. The ease with which the 
deficiency can be detected makes the screening of large numbers of individuals possible. 
The hemolytic anemia resulting in affected individuals upon exposure to certain drugs 
spawned the establishment of the field of pharmacogenetics, since it suggested that 
varied responses to drugs among individuals could be due to variability in other body 
enzymes. 

The use of G6PD as a marker was an invaluable tool in providing evidence sup- 
porting the hypothesis of X-chromosome inactivation. The fact that clones grown from 
individual cells from a heterozygous subject for G6PD variants exhibited only one type of 
G6PD enzyme indicated that only one of the tvi'o X chromosomes was active in an indi- 
vidual cell. These findings led to research on the origin of neoplasms and origin of 
atherosclerotic plaques and provided strong evidence for the monoclonal origin of most 
tumors. Finally, the study of G6PD deficiency may be an important tool in determining 
enzyme structure -function relationships. Over 100 variants of G6PD have been sho'wn 
to exist and the majority of these are probably the result of a single substitution of an 
amino acid. As the amino acid sequence and three-dimensional structure of these 
variants will be determined, the relationship of these amino acid substitutions to the 
activity of the enzyme could yield important information regarding the detailed mechan- 
ism of enzyme action. 

This case represents a reverse pattern from the other cases studied during the 
research program for the President's Biomedical Panel. Rather than progressing from 
bench to bedside, the development of knowledge about G6PD deficiency has gone from 



Q-2 

bedside to bench. The case started with the clinical observation of anemia resulting 
from administration of certain types of drugs several years ago. At present, the results 
of the research on G6PD deficiency are not utilized in a therapeutic method for treating 
patients, but are utilized in basic studies on human genetics, carcinogenesis, athero- 
sclerosis, and enzyme biochemistry. This work, in turn, is likely to affect therapeutic 
and preventative advances in these fields which are quite unrelated to their research 
origin. The study of G6PD shows the interrelation of the different branches of biology, 
biochemistry, genetics, and medicine, and how important advances are derived from 
unexpected quarters. 



HISTORICAL ACCOUNT 



The first documented report of drug-induced hemolytic anemia was in 1926 by 
Cordes, who noted the effect in a patient who had been administered the antimalarial 
synthetic drug Pamaquine. The potential seriousness of drug-induced hemolytic anemia, 
however, was not recognized until the end of World War II. At the start of the war, the 
seizure by Japan of the primary sources of quinine prompted an intense search for other 
antimalarial drugs, since the South Pacific Theatre was an area where malaria was 
endemic. Over 16,000 compounds were screened for antimalarial activity and a number 
of potential drugs v^ere identified including Atabrine. In 1944, a group was formed at 
the University of Chicago in cooperation with the Army Medical Research Unit to study 
antimalarial drugs. In these studies, the group encountered the hemolytic anemia in- 
duced by several of the antimalarial drugs, and these findings launched a large-scale 
effort to determine the cause of the anemia. In 1948, Earl reported that Pamaquine 
caused hemolytic episodes in 10 to 15 percent of American blacks but only rarely in 
Caucasians. In 1951, the group began a concerted effort to study the mechanism of 
hemolysis in order to develop safer antimalarial and other therapeutic agents. It was 
noted in 1952 by Hockv/ald that the antimalarial drug Primaquine also caused hemolytic 
anemia, and after this report, the term "Primaquine sensitivity" became a synonym, for 
susceptibility for drug-induced hemolytic anemia. 

The group at the University of Chicago was composed of many young, enthusiastic 
researchers, and in the period from 1952 to I960, a host of papers elucidating the 
mechanism of hemolytic anemia emanated from this group. Studies on the mechanism 
of drug-induced hemolytic anemia were greatly aided by the cooperation of prisoners 
from the penitentiary at Joliet. Since there was no animal model for the drug-induced 
hemolytic anemia, the accessibility to human subjects was a vital factor in the progress 
■which was made. Dern and co-workers in 1954 noted that Primaquine -induced hemolysis 
was self-limited; that is, after the initial hemolysis, the susceptibility of red cells to 
further destruction by repeated doses of the drugs v/as greatly reduced. They then began 
a systematic study to determine whether the cause of the hemolysis was due to a factor 
external to the red cell or was within the cell itself. To do this, a series of experiments 
utilizing Cr^l-labeled red cells was begun. The background for these experiments was 
suggested by earlier work which had demonstrated that sickle cell anemia was an intra- 
cellular defect. In Bern's experiment, the prison volunteers were divided into two 
groups: those who were sensitive to Primaquine and those who were not sensitive. Red 
cells were removed from sensitive subjects, labeled with Cr^^, and reinfused in insensi- 
tive subjects. The insensitive subjects were then given Primaquine and analysis of their 
blood samples showed that their own cells did not hemolyze. However, the infused cells 
from Primaquine -sensitive patients were hemolyzed. In a second series of experiments, 
red blood cells were removed from Primaquine-insensitive subjects, labeled with Cr^l, 
and reinfused in sensitive subjects. After a hemolyzing dose of Primaquine was given 



Q-3 

to the sensitive subjects, the results indicated that the cells from the insensitive sub- 
jects v^^ere not hemolyzed while the sensitive subjects' own cells were hemolyzed. These 
cross-transfusion experiments showed that the defect causing sensitivity to Primaquine 
and related drugs was intrinsic to the red cells themselves and not to a plasma factor. 
Bern's studies were followed by those of Beutler in the same group in 1954 who per- 
formed a series of cohort-labeling experiments with Fe^"^ These studies showed that 
older cells were more sensitive to drug-induced hemolysis than younger cells. This 
finding helped explain in part why the hemolysis was self-limiting. The initial dose of 
the hemolytic drug caused the destruction of the older cells which were then replaced by 
younger cells less susceptible to the effect of the drug. Beutler noted that the sensitive 
cells formed Heinz bodies (denatured globin within the red cells) while the nonsensitive 
ones did not (1954). He also noted that older cells have lower levels of certain enzymes 
than the younger cells and speculated that the increased hemolysis of the older cells may 
be related to the decreased enzyme levels. Studies of the mechanism of the hemolytic 
anemia were aided by Beutler's development in 1955 of an in vitro test for Primaquine 
sensitivity. In this test, red blood cells were incubated with acetylphenylhydrazine. 
Cells from Primaquine -sensitive individuals developed Heinz bodies while those from 
normal individuals did not. 

In further studies on red cells from Primaquine -sensitive individuals, Beutler et 
al. in 1955 found that the sensitive cells were similar to normal cells with respect to 
morphology, lack of sickling, Coombs' test response, acid hemolysis, methemoglobin 
production, and mechanical, chemical, and osmotic fragility. 

Beutler then began a systematic study of the metabolic path'ways in the Primaquine- 
sensitive red cells to determine the nature of the suspected biochemical lesion. He first 
examined the glycolytic pathway because of earlier work suggesting that hereditary 
spherocytosis might be a glycolytic defect. In a report published in 1955, Beutler could 
find no deficiency in any of the enzymes associated with the glycolytic pathway. In addi- 
tion, no deficiency in catalase, carbonic anhydrase, or choline esterase v/as found. He 
did find, however, that the levels of reduced glutathione were always lower than normal 
in sensitive cells exposed to Primaquine. Beutler's work was followed shortly thereafter 
by Carson et al. of the same group in 1956 who showed that the Primaquine- sensitive 
cells had a deficiency in glucose -6-phosphate dehydrogenase. Carson's studies were 
performed on hemolysates from red cells from normal Caucasians, normal blacks, and 
sensitive blacks. He found that the activities of glutathione reductase and 6-phospho- 
gluconic acid dehydrogenase were normal. With the finding of the lowered activity of 
G6PD and the lowered glutathione levels in sensitive cells, a partial picture of the 
mechanism of drug sensitivity began to emerge. Reduction of glutathione requires 
NADPH. The only source of NADPH in the red cell is the hexose monophosphate shunt, 
the first step of which is the oxidation of glucose-6-phosphate by G6PD. 

The second step in the hexose monophosphate shunt also produces NADPH. Thus, 
a deficiency in the first enzyme, G6PD, cuts off both sources of NADPH. When an 
oxidative drug such as Primaquine enters the red cell, glutathione is oxidized. Regen- 
eration of reduced glutathione depends upon the availability of NADPH. Since this latter 
compound is in short supply because of the enzyme deficiency in sensitive cells, the 
level of reduced glutathione falls. Glutathione perfoms several functions in red cells, 
among vi^hich is the protection of proteins from oxidation by providing free SH groups. 
The appearance of Heinz bodies is probably a direct result of the unavailability of re- 
duced glutathione. 

In addition to their basic work on the mechanism of Primaquine-induced hemolysis, 
the group at the University of Chicago also found that many other types of drugs besides 



Q-4 

the antimalarials could cause hemolysis. Among these drugs were antipyritics, anal- 
gesics, sulfonamids, sulfones, nitrofurans, and several miscellaneous drugs including 
chloramphenicol, trinitrotoluene, and vitamin K derivative s (Dern, et al. , 1955). In 
1957, Beutler developed a glutathione instability test as a method of detecting 
Primaquine -sensitive red cells and also as a method of screening drugs for hemolytic 
activity. In this test, the level of glutathione in sensitive cells dropped much more 
rapidly than that of normal cells upon exposure to a hemolytic drug. 

The link between the sensitivity to fava beans seen in Mediterranean populations 
and Primaquine sensitivity was established in 1957 by Sansone and Segni who found that 
patients with fava bean sensitivity also were sensitive to Primaquine. In the following 
year, Childs et al. at Johns Hopkins dennonstrated that Primaquine sensitivity is 
genetically determined. Childs used the glutathione instability test developed by 
Beutler as a marker for determining Primaquine sensitivity. He found that the familial 
pattern of G6 PD deficiency more closely resembled an X-linked trait than an autosomal 
trait. An X-linked genetic trait is passed from the mothers to daughters and sons and 
from the fathers to daughters only. Thus mothers and not fathers of some with the trait 
would manifest intermediate G6PD deficiency. Childs found that black males fell into 
two distinct classes of G6PD activity whereas females extended continuously from the 
lowest to the highest levels of activity. Proven female carriers fall into the group with 
intermediate levels. 

Further work on the mechanism of Primaquine sensitivity was continued at Chicago 
and by several other workers in the late 1950's. Marks et al. in 1958 found that G6PD 
as well as 6PGD (6 -phosphogluconate dehydronase) decreased in red cells as a function 
of age. This group also found that fava-sensitive patients had a G6PD deficiency (Gross, 
et al. , 1958). In further work, this group reported (Johnson and Marks, 1958) that 
Primaquine -sensitive red blood cells had a deficiency in the stimulation of the hexose 
monophosphate shunt by an oxidative compound, methylene blue. Using glucose l-C-*- , 
they showed that Primaquine -sensitive cells released significantly lower amounts of 
C-'-'*02 than did normal cells. They also showed subsequently that differences existed in 
G6 PD-deficient blacks and Caucasians. The enzyme in these two groups differed with 
respect to stability and activity (Marks, Banks, and Gross, 1959). Caucasians with 
G6PD deficiency in their red blood cells also had a reduced level of the enzyme in 
leukocytes. Affected blacks, on the other hand, had almost normal white blood cell 
levels of G6PD. The thermal stability of the enzyme in the red cells and white cells was 
lower in the affected Caucasians than in the affected blacks. This finding led them to 
suggest that a heterogeneity existed in genetically transmitted G6PD deficiency and led 
to a search by many other investigators for other variant types of the enzyme. 

Kirkman in 1959 purified G6PD from normal and from G6 PD-deficient blacks. He 
found the Michaelis constant, pH optima, and affinity for deoxyglucose-6 -phosphate were 
the same for both enzymes. In a subsequent paper (Kirkman, et al. , I960), he showed 
significant differences between the enzyme obtained from a G6 PD-deficient patient with 
congenital nonspherocytic hemolytic anemia and from that obtained from G6PD-deficient 
blacks. Investigation of the altered properties of the enzyme in G6PD-deficient subjects 
was continued by Marks, et al. , who in 1961 reported that the enzyme from certain sub- 
jects had a faster electrophoretic mobility than normal and a varied affinity for NADP. 
In 1962, Boyer, et al. , showed that the electrophoretic nnobility of G6PD from aifected 
blacks was 10 percent faster than normal G6PD. The rapidly moving enzyme was 
designated "A" and the normal "B". He further found that the enzyme in 20 percent of 
American blacks is A without exhibiting G6PD deficiency. Thus, 10 percent of male 
American blacks have a rapidly moving enzynne with G6PD deficiency (A-) and another 
20 percent of such males had an electrophoretical enzyme variant without enzyme 
deficiency (A+). 



Q-5 

After the report of Boyer, et al. , electrophoretic mobility- measurement became 
one of the key methods of characterizing variant G6PD enzymes. 

In the mid- I960' s, a great deal of work was done on the characterization of the 
enzyme and on the investigation of new G6 PD variants. Kirkman and Hendrickson in 
1962 showed that the enzyme was an aggregate of subunits. He also showed in 1964 that 
the G6PD from affected Caucasians had a similar electrophoretic mobility as normal B, 
yet utilized deoxyglucose -6 -phosphate and galactose -6 -phosphate relatively rapidly in 
comparison with the normal enzyme. In 1966, Yoshida developed a method of separation 
of G6PD enzymes from human blood which gave a high yield and purity. The next year 
he studied the black variant and found that it differed from normal G6PD in a single 
amino acid residue. In the A enzyme, aspartic acid was substituted for an asparagine. 

A host of new variants of G6PD was described during this period. Quite often, it 
was difficult to tell if the literature reports did indeed describe a new variant because 
of differences in the way in which the enzymes were characterized. In 196 7, the World 
Health Organization set up a committee which established definitive procedures for 
characterization of G6PD and served as a central point for verifying new G6PD variants. 
At the present time, over 100 variants have been reported and verified. Approximately 
20 of these cause relatively serious hemolytic anemia. Another 40 cause moderate to 
mild hemolytic episodes on exposure to certain drugs. In a number of cases, the 
severity of the hemolytic episodes could not be correlated with the degree of the G6PD 
deficiency. Yoshida in 1973 pointed out that this is an artifact resulting from the method 
used to measure G6PD activity in red cells. He showed that those variants associated 
with high hemolytic activity are strongly inhibited by NADPH and ATP. Nonhemolytic 
variants are not inhibited by NADPH and ATP. The ability of the hexose monophosphate 
shunt to produce NADPH is controlled by the physiologic conditions existing in the red 
cells. The conditions of the in vitro assay do not necessarily stimulate these conditions. 

The advances made in studying the molecular aspects of G6PD deficiency contrib- 
uted to a greater understanding of the clinical manifestations. Patients with a demon- 
strable G6PD deficiency can be placed in three categories. Patients in the first category 
have no clinical disorder even though their cells may demonstrate reduced G6PD activity. 
In the second group, patients have a hemolytic response with certain drugs but no chronic 
hemolytic disease. It should be pointed out that with subjects in this category, the 
response to drugs can vary. Patients with the Mediterranean type of deficiency are 
sensitive to a wider range of drugs and usually have more severe anemia than black 
patients. The third group of patients are those with congenital nonspherocytic hemolytic 
disease (CNSHD). Other enzyme deficiencies besides G6PD can cause CNSHD but G6PD 
deficiency is the largest single cause of the anemia. Patients in this group have chronic 
reticulocytosis and some experience chronic anemia. They also experience spontaneous 
hemolysis with no known cause, as well as hemolysis upon exposure to drugs and infec- 
tion. Some patients in this category experience juvenile cataracts. 

At the present time, there is no method of treatment for a patient with G6PD 
deficiency. This usually does not present a severe therapeutic challenge, however. The 
drug precipitating the hemolytic episode is simply discontinued and a nonhemolytic drug 
is substituted. If the subject is known to be G6PD deficient prior to administration of a 
therapeutic agent, the use of hemolytic drugs can be avoided. Many clinicians urge that 
patients of black or Mediterranean background be screened for possible G6PD deficiency 
before drug administration. 



Q-6 
Genetic Aspects 

One of the main benefits of the research in G6PD deficiency has been the use of 
this enzyme as a marker in the study of human genetics. The ease with which G6PD 
deficiency can be detected enables the screening of large numbers of subjects to trace 
familial patterns. As stated previously, Childs in 1958 showed that the deficiency was 
an X-linked genetic trait. Further work in the 1960's has shown that all cells in the 
affected individual contain the aberrant enzyme. The function of cells other than the 
nonnucleated erythrocytes is not compromised since nucleated cells continue to synthe- 
size enzyme throughout their lifetime. 

In I960, Motulsky suggested that G6PD deficiency may result from a selective 
advantage against falciparum malaria. He noted that the geographic distribution of G6PD 
deficiency coincided with the incidence of falciparum malaria and sickle cell hemoglobin. 
In the early 1960's, Motulsky and co-workers conducted several surveys of populations 
in Africa and in the Mediterranean area, and confirmed the hypothesis of a selective 
advantage against malaria which results from G6PD deficiency. They screened large 
numbers of subjects using a field test they developed based upon the decolorization of 
brilliant cresyl blue (Motulsky and Campbell-Kraut, 1961). The question naturally 
arises as to why G6PD deficiency should have a selective advantage. Incidence of G6PD 
deficiency in a given population certainly has some disadvantages such as neonatal 
hypobilirubinemia and hemolysis during acute infections. On the other hand, the 
enzyme deficiency may protect the subject from malaria. The malarial organism may 
need glutathione to survive, or the infected cell may be destroyed before the parasite 
can mature . 

It is difficult to demonstrate higher survival rates in G6PD-deficient subjects. 
However, Luzzatto, et al. , in 1969 produced experimental evidence of an increased 
resistance of G6PD-deficient cells against malaria. Using red blood cells of malaria- 
infected heterozygous G6PD-deficient -women who have t-wo cell populations, normal and 
G6PD deficient (see below), he showed that the deficient cells had fewer parasites than 
cells without the deficiency. Individual cells -were stained both for enzymatic activity 
and malarial parasites. 

The study of G6PD deficiency also spawned the field of pharmacogenetics. In 
1957, Motulsky prepared a special report on the relationship of untoward drug reactions 
to biochemical genetics for the Araerican Medical Association Council on Drugs. He 
noted that hyper susceptibility and hyposusceptibility to certain drugs might be traced to 
a genetically controlled variability of enzyme levels in the affected subjects. Citing 
G6PD deficiency as an example, he noted that the enzyme variation may be completely 
innocuous until the patient is presented with a certain drug. Enzymes or other proteins 
involved in the transport, metabolism, binding, or conjugation of the drug can be variant 
in different individuals and thus a varying response to the drug can result. In a further 
paper in 1964, Motulsky noted that if one plots a parameter of drug interaction such as 
rate of drug decay versus frequency in a normal population, the result usually is a uni- 
modal Gaussian distribution. A multimodal curve suggests variant enzyme levels in a 
given population. He gave several examples besides G6PD deficiency of variant enzymes. 
Among these are a variant response to succinyl choline caused by abnormal pseudo- 
cholinesterase, sensitivity of certain hemoglobins to drugs, and variations in dicou- 
marin metabolism. A World Health Organization report in 1973 cites many other exam- 
ples of altered response to certain drugs and discusses the public health aspects of 
pharmacogenetics. 



Q-7 

G6PD deficiency played a definitive role in the elucidation of the process of gene 
inactivation and in the demonstration of genetic mosaicism. In an X-linked chromosom.al 
trait only one expression occurs in hemizygous males because the mutant X gene is not 
opposed by :. normal allele. Full expression is rare in females because a homozygous 
female must inherit the mutant gene from both parents. Usually "affected"' women are 
heterozygous and carry one mutant X gene and one normal X allele. In 1961, Lyon 
suggested that in each somatic cell of mammals, only one of the two X chromosomes is 
active, so that on the average, 50 percent of cells have the paternal X chromosome and 
50 percent have the maternal X chromosome active. The progeny of each cell will 
represent the same active chromosome of the parent. Every female therefore is a 
mosaic. In a woman heterozygous for an X-linked trait, a portion of her cells would 
express the normal gene while others would express the altered gene. Beutler indepen- 
dently showed in 1962 that the red blood cells from heterozygous females displayed two 
components in the glutathione decay curve, thus indicating that two populations of cells 
existed. He discussed this point further in 1962 and used the hypothesis of gene 
inactivation to explain why the mothers of G6PD-deficient males exhibited a wide range 
of G6PD activity. Further evidence on the gene inactivation hypothesis came frora the 
work of Grumbach and Marks in 1962 who showed that the G6PD activity in human sub- 
jects was the same whether they had the XO or the XXXX complement of sex chromo- 
somes. The conclusion was that in each somatic cell, there was only one active X 
chromosome. 

Definitive evidence supporting the hypothesis of gene inactivation came from the 
work of Davidson in 1963. Using cultured fibroblasts from heterozygous G6PD-deficient 
females, he showed two different electrophoretic bands of G6PD. He then prepared sub- 
cultures from individual cells and found that in each subculture only one electrophoretic 
band of G6PD was exhibited. Some of the monocultures had the rapidly migrating band; 
others had the normal, slowly migrating band. These results demonstrated that only one 
gene was active on the X chromosome of each individual cell and that the progeny of that 
cell reflected the activity of the parent. 

These studies on the maintenance of a specific type of G6PD in cell lines prompted 
studies on the origin of tumors. H a tumor in a heterozygous woman arises from a 
single cell, then only one type of G6PD should be expressed in the tumor. If, however, 
the tumor arises from two or more cells, then the possibility exists that it will contain 
both types of enzyme. In 1965, Linder and Gartler studied uterine myomas in females 
heterozygous for two electrophoretic types of G6PD. They found that each tumor had 
only one type of enzyme, thus indicating a unicellular origin of the tumors. Fialkow, et 
al. , in 1967 used G6PD as a marker in chronic myolocytic leukemia and lymphosarcoma, 
and showed that these also originated from a single cell. Their results also had implica- 
tions about the origin of embryonic cell lines. They found that red blood cells and 
granulocytes were of one electrophoretic type, and concluded that they were from a com- 
mon stem cell. Many other types of neoplasia were investigated in the succeeding years 
(Kirkman review, 1971; Fialkow, 1974), and these indicated that most tumors were of 
unicellular origin while certain hereditary tumors were of multicellular origin. 

In addition to the studies in the origin of cancer, the use of G6PD as a marker also 
has provided insight into the origin of atherosclerotic plaques. Benditt and Benditt (1973, 
1974) studied 30 samples of arterial plaques from three subjects and 50 samples of non- 
plaque arterial intima from heterozygous female subjects. They found that the fibrous 
cap of each plaque was composed of cells producing only one type of G6PD. On the other 
hand, normal intimia and medial tissue had both types of the enzyme present. These 
studies suggest monoclonal origin of the cells in the atherosclerotic plaque. They 
speculated that these cells had the properties of a benign neoplasm. A mutagenic agent 



Q-8 

such as constituents of cigarette smoke, cholesterol, or a metabolite could have 
initiated the transformation from a normal intimal cell to the neoplastic state. If these 
data can be substantiated, they have important implications on the origin and prevention 
of atherosclerosis. 



REFERENCES 

Alving, A. S. , et al. , J. Clin. Invest., 27, 2 (1948). 

Benditt, E., Circ. , 50^, 650 (1974). 

Benditt, E. P., and Benditt, J. M. , Proc. NAS(US), 70, 1753 (1973). 

Beutler, E. , J. Lab. Clin. Med., 49, 84(1957). 

Beutler, E., in Mechanisms of Anemia in Man, I. Weinstein and E. Beutler (eds. ), 
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Beutler, E., Bern, R. J., and Alving, A. S. , J. Lab. Clin. Med., 44, 439 (1954). 

Beutler, E., Dern, R. J., and Alving, A. S. , J. Lab. Clin. Med., 45, 30 (1955). 

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Q-9 

Fialkow, P. J., etal., Proc. NAS(US), 58, 1468 (1967). 

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MotuLsky, A. G., JAMA, _1_65, 835 (1957). 

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Yoshida, A., Science, 129, 532(1973). 



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I Umks Ocmoniirgicd diflsroncsi ii 
G6P0 dolicieni biacki and 

MR/D/U/G/M 
) Moiuliky SuMOiiPd G6P0 ctol/eia' 



1961 Lyon Hypolha)! 



1962 Bcullir Raloiod iprgad ol G6P0 aciiv 
ily In haioroivoout woman K 
Bonollc motalclim 

MR/S/U/G/M 

1962 eeuilar Shonad Ino convonanii ol 
GSH decay curve lo holei- 

MR/D/U/G/M 

1962 Grumbacti Slion«J liurc GSPO gciivl 

In Hibiccti liauirifl nida 
ranjB d< X comolonwrn ol 

NR/D/U/G/M 

1963 Davidian Oalinllive nldence of gene 



MR/D/U/G/M 

1960 Kirkmon Puiili8dG6PD (rom Mtvenjl 

la-jices and ihowed dillor. 

MR/S/U/G/M 

1961 Maiki Studied GGPD f'Om normal and 

MR/S/U/G/M 
1961 Moiuliky O0ve[oped fii 



)' G6PD ai 






MR/S/U/G/M 
in ShDHCd G6P0 >vai eggit-^ 
NR/S/U/G/M 



MR/O/U/G/G 



1967 Fialkow ShOMd monoclonol origin 
MR/S/U/G/M 




1962- Sim. 



nlhcroiclaroilc plaquei uil 
G6P0 marker 

MR/S/U/G/M 



MR/S/U/G/M 



1967 YoihldaShow 



. Aiparsgina changod ic 
MR/D/U/G/M 



HISTORIOGRAPH - GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY 



1964 Motuliky Furihei defined Irald 
pharmocOBOnBiiei 
MR/S/U/G/M 



R-1 
APPENDIX R. ALPHA- FETOPROTEIN 

INTRODUCTION AND SUMMARY 



Alpha- fetoprotein (AFP) is one of the principal glycoproteins found in embryonic 
life and is synthesized by fetal liver cells. The biological function of this molecule has 
not been defined, but many investigators have noted the resemblance of AFP and serum 
albumin in physicochemical and some physiological properties, AFP production in the 
fetus is inversely related to serum albumin production, with AFP being barely detected 
in the serum at birth, and not present at all during adult life, 

AFP is not synthesized by normal adult liver cells; however, if these cells 
transform to cancer cells, synthesis of this protein is resumed. This resynthesis is 
easily detected in the serum of patients with hepatoma and has provided a successful 
supplemental diagnostic tool for the detection of the tumor-bearing patient. Other liver 
diseases such as infectious hepatitis, cirrhosis, and metastatic cancer of the liver have 
been associated with reappearance of AFP in the patient's serum, but none with the 
substantial positive correlation found with hepatoma. 

Recently AFP has been measured in maternal serum and prenatal amniotic fluid, 
and it has been found that AFP concentration was an excellent marker of gestational age 
of the fetus. Quantifications of AFP during pregnancy also detected fetal distress 
problems and certain defects of neural- tube development. 

This report will deal with AFP in four areas of experimentation, as outlined in 
the Historiograph. First, the discovery of a fetal specific protein in 1944 and its 
characterization through the next 3 years; second, the association of this fetal protein 
with cancer of the liver, its use as a diagnostic tool, and its possible use as an innmuno- 
therapeutic agent; third, the quantification of AFP during pregnancy for the detection 
of defects in neural- tube development; and finally, the most recent observation that 
AFP is able to inhibit the immune response. Overlaying these investigations increasing 
our knowledge of AFP is the development of miore sensitive techniques for its detection. 
Knowledge about this protein has expanded rapidly with this better technology, as 
evidenced by the increase in published articles on AFP from 160 before 1970 to well 
over 300 after 1970. 



HISTORICAL ACCOUNT 



The first fetal specific protein, fetuin, was discovered by Pederson in 1944 
in fetal calf serum. This observation gave substantive birth to the concept that there 
are unique substances in the fetus which are absent in the normal adult. Fetuin, 
however, is different from AFP, but this was not completely defined until 1968 when 
definitive immunochem.ical data were supplied from Czechoslovakia by Kithier et al. 
In 195 6, Bergstrand and Czar reported the finding of a new fraction in the electro- 
phoresis of plasma proteins migrating between the albumin and alpha- one regions in 
a few- week's- old fetus which was not present in maternal serum. 



R-2 

In 1966 David Gitlin of the United States arbitrarily labeled this protein alpha- fetoprotein 
and the label was adopted by the International Agency on Research on Cancer in 1969. 

Basic understanding of AFP in normal biology was not advanced after its discovery 
in 1956 until 1967 when Gitlin and Boesman showed that AFP is synthesized in the liver, 
more specifically by the hepatocytes. Their experiments consisted of perfusing various 
fetal organs with radio- labeled amino acids and examining the organs for .incorporation 
of the labeled amino acid into newly synthesized AFP, They showed no incorporation, 
except for the liver, in spleen, kidney, lung, placenta, intestine, stomach, heart, or 
brain. These investigators also showed during the same year that AFP was a common 
protein found in the fetal serum of at least 18 mammalian species. 

Many investigators contributed to the physicochemical characterization of AFP, 
but the most definitive work -was performed in Japan and culminated in a publication in 
1970 by Nishi, The work reported by Nishi sho-wed that AFP found in serum -was identical 
to that found in the serum of patients with hepatoma. In a companion report the same 
year with Hirai, he also assemibled the cumulative knowledge on physicochemical 
characteristics of AFP. They showed that AFP was a distinct chemical entity. In 
1971, Alpert et al, described different forms of AFP, The differences were due to 
varying amounts of sciatic acid associated with each molecule; the two different forms 
of AFP, however, are immunochemically identical. 

In 1971 Ruoslahti and Seppala developed a radioimmunoassay for the detection of 
AFP. This new sensitive technique allowed the precise measurement of AFP in amniotic 
fluid, and these Finnish investigators reported in 1972 that AFP in the amniotic fluid 
reflected the gestational age of the fetus. AFP was found in highest concentration 
during the first trimester, peaking at about 12 weeks and gradually decreasing there- 
after until birth. This observation laid the groundwork for the use of AFP levels in 
amniotic fluid for the detection of fetal neural- tube defects, 

A possible function of AFP was introduced in 1972 by Uriel et al, from France 
by their finding the estrogen-binding properties of AFP. Using cell affinity labeling with 
tritated estrogens, they found that the transitional cells (i,e, , AFP-producing cells) 
were labeled the nnost. The reaction was specific, as no binding could be demonstrated 
for testosterone, progesterone, cortisone, or dexamethasone. The significance of this 
finding has yet to be determined. 



AFP in Cancer 



In 1932 Hirszfeld and his co-workers in Germany postulated that a fetal- specific 
substance must be associated with malignant- tumor tissue. The bridge between the 
above postulate and actual observation was made by Abelev and his co-workers in 
Russia in 1963, They observed an "alpha em.bryo- specific antigen component" in the 
sera of adult mice with primary cancer of the liver. The following year, also in 
Russia, TatariQOv detected a similar substance in the sera of two patients with 
hepatonna. These observations set the stage for many clinical studies using AFP 
as a possible tumor-specific antigen. It was hoped that this protein might be used 
to detect previously unsuspected cancer and thus becomie a potent diagnostic tool. 



R-3 

AFP was found in the serum of patients with teratoblas toma of the testis by Abelev in 
1967, but not in other embryonal tumors. The most significant study occurred in 1970, 
led by O'Connor et al. under the auspices of the World Health Organization, where this 
group attempted to determine the value of AFP as a detector of hepatoma. They 
collected serum samples from all over the world from patients with hepatoma. A great 
majority of these cases were biopsy- proven hepatomas. Their results showed that the 
positive finding of AFP in the sera was greatly dependent upon the method used for 
detection. Forty-five percent of the sera were positive if assayed by immunodiffusion, 
while over 85 percent were positive when assayed by the more sensitive radioimmuno- 
assay technique. With application of more sensitive techniques for the detection of AFP, 
the use of AFP as diagnostic tool has been greatly modified by finding other liver disease 
associated with AFP positive tests. This has led some investigators to consider the 
AFP assay as cancer- distinctive rather than cancer- specific, since false positive re- 
sults were obtained in cases of hepatitis and cirrhosis. Another reason is that AFP 
levels may be elevated in tumors other than those derived from the liver, e. g. , gastric 
cancer; colonic, pancreas, and prostatic cancer; and metastatic liver disease, as well 
as teratoblastomas. 

The next significant observation regarding AFP in cancer was suggested by many 
investigators but was first reported by Purtilo et al. in 1973, when they demonstrated 
the efficacy of AFP levels in monitoring the response of hepatoma patients to surgical 
resection, irradiation, and chemotherapy. The loss of AFP from the serum indicated 
a positive response to therapy, whereas persistent or rising AFP levels indicated non- 
responsiveness. 

One of the areas of AFP's role in cancer that remains largely unexplored but 
may offer a mode of specific cancer therapy is the use of anti-AFP sera to retard the 
growth of hepatoma cells. In 1974, Tsukada et al. of Japan observed that anti-AFP 
slowed the growth of cultured hepatoma cells. They found that the antisera adhered to 
AFP secreted on the cell surface and postulated that these antigen antibody complexes 
inhibited cell multiplication. 



AFP as a Marker of Abnormal Fetal Development 

In 1972, Ruoslahti and Seppala of Finland found that there was an increased prob- 
ability of abortion after 13 weeks of pregnancy in those mothers with high AFP levels 
in their serum. This suggested to Brock and Sutcliffe of England that AFP levels 
which were abnormal during gestation might be used to detect defective fetuses. 
They reported in the same year that AFP was a good antenatal diagnostic tool for 
the prediction of anencephaly and spina bifida. This observation has been confirmed by 
numerous investigators. In 1974, Seppala and Unnerus described the first case of ele- 
vated AFP levels in amniotic fluid of a fetus with hydrocephalus. The diagnostic uses 
of AFP in pregnancy are probably miore precise than those of AFP in cancer. 



R-4 
AFP as an Immunosuppressive Agent 



In 1969, Hull et al. in the United States observed that in chemically induced 
hepatomas of monkeys, the early tum.or tissue showed a prominent lymphocytic infiltrate. 
This infiltrate disappeared later in the disease and could be correlated with the rise in 
production of AFP. This finding suggests that AFP as a product of the tumor tissue may 
have some immunosuppressive properties. 

In 1974, Waldman and Mclntire reported elevated levels of AFP in patients with 
ataxia- telangiectasia. This disease is characterized by an immunodeficient state, the 
absence of gam.ma globulin, and recurrent infections. These observations also suggest 
that AFP might play a role in causing this immunosuppressed state. 

A possible role for AFP in suppressing the immune response was reported in 1975 
by Murgita and Tomasi of the United States. In one series of experiments, both mouse 
amniotic fluid containing AFP and isolated AFP acted as noncytotoxic inhibitors of the 
primary cellular synthesis of IgM antibody in splenic plaque- forming cells to sheep 
red- blood cells and the secondary response of IgM, IgA, and IgG synthesis. In a second 
series of experiments AFP was shown to suppress thymus derived T- cell- dependent 
functions of allogenic and mitogen- induced lymphocyte transformation These findings 
have wide implication concerning the biology of AFP in tumor immunology and specific 
cancer therapy and obviously deserve more aggressive experimentation in areas of 
AFP as an immunosuppressive. 



REFERENCES 



Abelev, G, I,, Cancer Res, , ]A_, 295 (1971). 

Abelev, G. I,, Cancer Res. , 2£, 1344 (1968). 

Abelev, G. I. , Protides of the Biological Fluids, 2_8, 203 (1970), 

Abelev, G. I. , Alpert, E. , Hull, E. W, , Masseyeff, R, , de Nechaud, B, , 
Tatarinov, Y. , and Uriel, J., Bull. Org. mond. Sante, 43_, 311 (1970). 

Abelev, G. I, , Assecritova, I. V. , Kraevsky, N. A. , Perova, S, D. , and 
Perevodchikova, N, I, , Int. J. Cancer, 2, 551 (1967). 

Alpert, E. , New Eng. J. Med., 290, 568 (1974), 

Alpert, E, , Schur, P, , Drysdale, J, , and Isselbacher, K, , Fed, Proc, , 30, 
246 (1971). 

Bergstrand, C, G. , and Czar, B. , Scand, J, Clin, Lab, Invest, , 8, 174 (1956), 

Brock, D, J, H, , and Sutcliffe, R, G, , Lancet, 2, 197 (1972), 



R-5 

Day, E. D. , The Immunoc'hemistry of Cancer, C, C. Thomas (Ed, ), Springfield, Illinois, 
170 (1965). 

Gitlin, D, , and Boesman, M, , Comp. Biochem. Physiol, , 21, 327 (1967). 

Gitlin, D. , and Boesman, M, , J. Clin, Invest, , 45, 1826 (1966), 

Gitlin, D. , and Boesman, M. , J, Clin, Invest, , 46, 1010 (1967). 

Hirszfeld, L. , Z. Immun. Forsch. Exp. Ther, , 64, 81(1929). 

Hull, E. , Carbone, P. , Gitlin, D. , O'Gara, R. , and Kelly, M. , J. Natl. Can. Inst. , 
42, 1035 (1969). 

Kew, M. , Gut, 15_, 814 (1974). 

Kithier, K. , Masopust, J, , and Radl, J, , Biochim, Biophys, Acta, 160 , 135 (1968). 

Lau, H. L. , and Linkin, S. E. , Am. J. Obstet. Gyn. (to be published). 

Lawrence, D. J. R. , and Heville, A. M. , Br. J. Can., 26_, 24 (1972). 

Murgita, R. A, , and Tomasi, T. B. , J. Exp. Med. , 141 , 269 (1975); ibid, J. Exp. 
Med. , 141 , 440 (1975). 

Nishi, S. , Cancer Res. , 30, 2507 (1970). 

Nishi, S. , and Hirai, H. , Protides of the Biological Fluids, ]_8, 43 (1970). 

Nishi, S. , Watanabe, H. , Tsukada, J. , and Hirai, H. , Protides of the Biological Fluids, 
18_, 43 (1971). 

O'Connor, G. T. , Tatarinov, Y. S. , Abelev, G. I. , and Uriel, J. , Cancer, 25, 
1091 (1970). 

Pederson, K. , Nature, 154 , 575 (1944). 

Purtilo, D. T. , Kersey, J. H. , Hallgren, H. M. , Fox, K. R. , and Yunis, E. J. , 
Am. J. Clin. Path. , 59_, 295 (1973). 

Purtilo, D. T. , and Yunis, E. J., Fed. Proc. , 3_0, 634(1971). 

Ruoslahti, E. , and Seppila, M. , Nature, 235 , 161 (1972). 

Ruoslahti, E. , and Seppala, M. , Int. J. Can. , 8, 374 (1971). 

'Seppala, M. , Bagshawe, K. D. , and Ruoslahti, E. , Int. J. Can., J^, 478 (1972). 

Seppala, M. , and Ruoslahti, E. , Br. Med. J., 4, 769 (1972). 
Tatarinov, Y. S, , Vopr. Med. Khim. , 1_0, 584 (1964). 



R-6 

Tatarinov, Y. S. , Fed. Proc. (Trans. Suppl. ), 25_, 344 (1966). 

Tsukada, Y. , Mikuni, M, , Watabe, H, , Nishi, S, , and Hirai, H. , Int. J. Can, , 
13_, 187 (1974). 

Uriel, J. , de Nechaud, B. , and Dupiers, M. , Biochem, Biophys. Res. Comm, , 46, 
1175 (1972). 

Uriel, J., de Neuchaud, B. , Stanislawski-Birenchwaig, M. , Masseyeff, R. , 
Leblanc, L. , Quenum, C. , Loisillier, F, , and Grabar, P., Presse Med., 76, 
1415 (1968). 



HEPATOMA) 



cific 
r tissue 
II 



otein in 
with 



l/M 



proteins 
IS with 



/M 



n asso- 

:toma 

/M 



IMMUNOSUPR 



1969 Hu" '"h"", 



ISOLATION AND CHARACTERIZATION A DETECTOR OF CANCER (HEPATOMA) 





CODES 


NR 


Nonmission-Oriented Research 


MR 


Mission-Oriented Research 


DV 


Development 


S 


Significant Event 


D 


Decisive Event 


U 


U.S. Event 


w 


Foreign Event 


G 


Government Funding 


P 


Private Funding 


M 


IVIedical School - Hospital - 




University 


1 


Research Institution 


GL 


Government Laboratory 


C 


Company 



1944 PerefSOft Discovery of fetal tpecific pro- 
tein in calf (Brum - fetuin 
NR/S/W/G/M 



1956 Bergslrand Oiscovery of tetal prqtein 
jn human serum 

NR/D/W/G/M 



IMMUNOSUPPRESSIVE PROPERTIES 



1969 Hull Chemicellv induced tumors 

(hepatoma) in monkeys: showed 
disappearance of lymphocytic in- 
filtrate in tumor tissue correlated 
with rise in production of alpha- 
fetoprotein 

NR/S/U/G/M 



1974 Waldman Increased serum alpha-fe 
protein levels in immunt 

WIR/S/U/G/M 



a Immunosuppressive propertie 
of alpha-tetoprotein in mice. 
MR/D/U/G/IVl 



1967'Giltin - Fetal specific protein in 18 
different mammalian species 
— Localization of alpha-feto- 
protein production in liver of 
letus, 

NR/S/U/G/M 

1968 Kilhier Alpha-fetoproiein different 
from fetuin 

NR/S/W/G/M 

1970 Nishi Physicochemical characterization 
of alpha-fetoprotein from tetal 
serum and hepatoma patient 



MR/D/W/G/M 



A DETECTOR OF ABNORMAL 
FETUS IN PREGNANCY 



1971 Alpert Discovery of different forir 
alpha-feioprotein 

NR/0/U/G/M 



a Found increased chance of 
abortion alter 13 weeks. 
Gestation with increased alpha- 
feioprotein levels in mother. 
MR/D/W/G/M 



1972 Brock Alpha-fetoprotein in the anten: 
diagnosis ol anencephaly and 
spina bifida 

MR/D/W/G/M 



1974 Seppala 1st case of elevated alpha-feto- 
protein in amniotic fluid of 
fetus with hydrocephalus 
MR/S/W/G/M 



1972 Seppala Measui 



!ment of alpha-feto 
during pregnancy 
MR/D/W/G/M 



1972 Uriel Estrogen binding by alpha- 
fetoprotein 

MR/S/W/G/M 



1932 Hirsifeld Postulates fetal-specific 

substance in cancer tissue 
NM/S/W/G/ 



1963 Abelev Discovery of fetal protein in 
serum ol adult mice with 
hepatoma. 

NR/S/W/G/M 



1964 Tatarinov Discovery of fetal proteins 
in serum of humans with 
hepatoma. 

NR/S/W/G/M 



V Fetal protein in serum asso- 
ciated with teratoblastoma 
MR/S/W/G/M 



1970 O'Connor Collaborative study showed 

85% serum positive tor alpha- 
fetoprotein in patients with 
known hepatoma. 
MR/D/W/G/I 



IMPROVED METHOD FOR QUANTITATIVE 
DETECTION 



Alpert Application o( counterimmuno- 
eleclrophoresis tor detection of 
alpha-letoproteln in serum. 
DV/S/U/G/M 



1973 Purtilo Use of alpha-tetoproteln to 

monitor lesponse of hepatom 
to surgical resection irradiatio 
and chemotherepy. 

MR/S/U/P/M 



1974 Tsukada Show growth of cultured hepa- 
toma cells with anti-alpha- 
letoprotein 

MR/S/W/G/M 



1971 Ruoslahti Development ol radioimmuno- 
assay lor detection ol alpha- 
fetoprotein in serum and 
amniotic fluid 

DV/S/W/G/M 



1975 World Health STD, 72/225 established. 

Organization lor reference of alpha- 
fetoprotein. 
DV/D/ / / 



HISTORIOGRAPH - ALPHA-FETOPROTEIN 



S-1 

APPENDIX S. PRIOR STUDIES OF 
RESEARCH AND INNOVATION 



Listed below are a number of studies of research and of the innovative process as 
examples of the kind of background infornnation on protocol and methodology available 
to this program. 

'An Assessment of the Role of Research Involving Living Human Fetuses in Advances 
in Medical Science and Technology", Final Report to the National Commission for the 
Protection of Human Subjects, Contract NOl -HU-5-2 12Z, from Battelle, Columbus 
Laboratories, March 28, 1975. 

"Interactions of Science and Technology in the Innovative Process: Some Case Studies", 
Final Report to the National Science Foundation, Contract NSF-C-667, from Battelle, 
Columbus Laboratories, March 19, 1973. 

"Invisible Colleges: Diffusion of Knowledge in Scientific Comnnunities", Crane, Diana, 
University of Chicago Press, Chicago, 1972. 

"Organizing for Innovation: A Systems Approach to Technical Management", Morton, 
J. R. , McGraw-Hill, New York, 1971. 

"Project Sappho: A Study of Success and Failure in Innovation", Achilladelis, B. , 
Jervis, P. , and Robertson, A. , Science Policy Research Unit, University of Sussex, 
Brighton, England, 1971. 

"Science, Technology, and Society in Seventeenth Century England", Merton, R. K. , 
Fertig, New York, 1970. 

"The Social Environment for Sustained Technological Gro-wth", Pool, I. deS., pp 269- 
282 in Cetron, M. J. and Goldhar, J. D. , eds. , The Science of Managing Organized 
Technology, Gordon and Breach Science Publishers, Inc., New York, 1970. 

"Successful Industrial Innovations", Myers, S. and Marquis, D. G. , NSF 69-17, U. S. 
Government Printing Office, Washington, 1969. 

"Technological Forecasting and Long-Range Planning", Ayres, R. U. , McGraw-Hill, 
New York, 1969. 

"The Structures of Publication in Science and Technology", Price, D. J. deS. , pp 91- 
104 in Gruber, W. H. and Marquis, D. G. , eds. , Factors in the Transfer of Tech- 
nology, M. I. T. Press, Cambridge, 1969. 

"Applied Research, Definition, Concept and Themes in Applied Science and Technolog- 
ical Progress", Brooks, H. , report to the U. S. National Academy of Sciences to the 
Committee on Sciences and Astronautics, U. S. House of Representatives, Washington, 
D. C, 1967. 

"Communications in the Research and Development Laboratory", Allen, T. J. , Tech- 
nology Review, 70, 30-37, 1967. 

"Technology and Change", Schon, D. A. , Dell, New York, 1967. 



S-2 

"Scientists in Organizations", Peltz, D. C. and Andrews, F. M. , Wiley, New York, 
1966. 

"The Scientific Community", Hagstrom, W. O. , Basic Books, New York, 1965. 

"Little Science, Big Science", Price, D. J. deS. , Columbia University Press , New 
York, 1963. 

"Patterns and Problems of Technical Innovation in American Industry", Arthur D. 
Little, Inc., NSFC-65344, A. D. Little, Cambridge, 1963. 



T-1 
APPENDIX T. GLOSSARY =;= 



ABO Blood groups: usually designates the four groups of individuals — A, B, O, and 
AB — identified by the ABO system discovered by Landsteiner. Other systems, 
like Rh, are said to define blood types, rather than groups. Each blood group 
or type is based on a blood factor, or antigen, that is present in its members' 
red cells . 

ABO-incompatible: one person is ABO- incompatible to another if his red cells carry 
A and/or B antigen that will provoke an immune reaction if transfused into the 
other, owing to the presence in the other's serum of anti-A and/or anti-B 
antibody. 

adrenergic: activated by, or characteristic of, secreting epinephrine or substances 
with similar activity; the term is applied to those nerve fibers that liberate 
norepinephrine at a synapse when a nerve impulse passes, i.e., the sympathetic 
fibers . 

agglutinin: antibody which aggregates with a particular antigen, e.g., bacteria, follow- 
ing combination with the homologous antigen in vivo or in vitro. 

aglycone: the noncarbohydrate group of a glycoside molecule. 

akinesia: absence, loss, or impairment of voluntary activity. 

albumin: a protein that is soluble in water and coagulable by heat; found in most animal 
and many vegetable tissues. 

allergenic: acting as a substance capable of inducing allergy or specific hypersensi- 
tivity; such a substance may be a protein or a nonprotein. 

amniocentesis: passage of a hollow needle into the amniotic fluid that surrounds the 
fetus in the womb, and the removal of a specimen of the fluid. 

amnion: in placental mammals, the thin but tough extraembryonic membrane that con- 
tains the fetus and amniotic fluid. 

amniotic fluid: fluid within the amniotic sac surrounding the fetus. 

analgesic: relieving pain; the agent that alleviates pain without causing loss of 
consciousness . 

anamnestic: the capability of being recalled. 

anaphylactic shock: a violent attack of symptoms produced by a second injection of 

serum or protein and due to anaphylaxis (an unusual allergic reaction to foreign 
protein or other substances). 



androgenic: producing masculine characteristics. 



' Adapted primarily from Dorland's Illustrated Medical Dictionary , 25th ed. , W. B. Saunders Co. , Philadelphia (1974). 



T-2 

anemia: a deficiency state of blood in which there are too few red cells or the red cells 
are of too poor quality to fulfill their oxygen- transporting function in the body. 

anencephaly: congenital absence of the cranial vault, with cerebral hemispheres com- 
pletely missing or reduced to small masses attached to the base of the skull. 

anesthesiology: study of the loss of feeling or sensation, especially pain, as it is 
induced to permit performance of surgery or other painful procedures. 

angina pectoris: a spasmic thoracic pain, with a feeling of suffocation and impending 
death. 

angiocardiography: roentgenography of the heart and vessels after introduction of con- 
trast material into the heart and blood vessels. 

anorexia: lack or loss of the appetite for food. 

antecubital: situated in front of the elbow. 

Antergan: trademark for a preparation of phenbenzamine. 

anthelmintic: destructive to worms. 

anthrax: an infectious disease of ruminants resulting from the ingestion of spores of 
Bacillus anthracis in the soil, often leading to sudden death. Transmittable to man. 

antibiotic: a chemical substance produced by a microorganism which has the capacity, 
in dilute solutions, to inhibit the growth of or to kill other microorganisms. 

antibody: a substance produced in the body in response to a specific foreign material, 
or antigen. An antibody acts, in an immune reaction, to defend the body by 
destroying or nullifying the antigen against which it is made. 

antiedemic: an agent that prevents or alleviates the presence of abnormally large 
amounts of fluid in the intercellular tissue spaces of the body. 

antigen: a substance on or in a red cell that is antagonistic to a human or other 

organism in such a way that it forms an antibody against it. The blood factors 
A, B, and Rh are antigens. Chemically, most antigens are proteins. 

antihistimine: a drug that counteracts or reduces the physiological effects associated 
with histamine production in allergies and colds. 

antimetabolite: a substance bearing a close structural resemblance to one required for 
normal physiological functioning, and exerting its effect by interfering with the 
utilization of the essential metabolite. 

antimicrobial: killing microorganisms, or suppressing their multiplication or growth. 
Also, an agent that kills microorganisms or suppresses their growth. 

antiovulation: suppression of the discharge of a secondary oocyte from a vesicular 
follicle of the ovary. 

antipyretic: an agent that relieves or reduces fever. 



T-3 

antiseptic: preventing decay or putrefaction; a substance that will inhibit the growth 
and development of microorganisms without necessarily destroying them. 

antiserum; the clear portion of liquid that contains antibodies; it may be obtained from 
an animal that has been immunized either by injection of antigen into the body or 
by infection with microorganisms containing the antigen. 

anuria: absence of excretion of urine from the body. 

aortic stenosis: narrowing or stricture of the aorta. 

ASD: atrioventricular septal defect. 

atherosclerotic: pertaining to a common form of hardening of the arteries which 

deposits yellowish plaques containing cholesterol and lipoid material within the 
arteries . 

atrioventricularis communis: a congenital cardiac anomaly in which the cushions 
within the heart fail to fuse, the atrioventricular canal is undivided, a single 
atrioventricular valve has anterior and posterior cusps, and there is a defect of 
the membranous interventricular septum. Also called persistent common 
atrioventricular canal. 

attenuated: thinned; weakened; rendered less virulent. 

Aureomycin: trademark for preparations of crystalline chlortetracycline hydrochloride. 

autosome: any ordinary paired chromosome as distinguished from a sex chromosome. 

A-V: atrioventricular; arteriovenous. 

avian malaria: malaria of birds due to numerous species of the genus Plasmodium, 
and these protozoa are transmitted by mosquitoes. 

azotremia: an excess of urea or other nitrogenous bodies in the blood. 

barbital: chemical formerly used as a long-acting hypnotic and sedative. 

barbiturate: a salt or derivative of barbituric acid; causing chills, headache, fever, 
and cutaneous eruptions. A sedative or hypnotic. 

beta-lipoprotein (low density): a plasma protein containing relatively more cholesterol 
and triacylglycerides and less protein than high-density alpha- Lipoprotein. 

biomedical: pertaining to the application of the natural sciences (biology, biochemistry, 
biophysics, etc.) to the study of medicine. 

biopsy: removal and examination, usually microscopic, of tissue from the living body, 
performed to establish precise diagnosis. 

bilirubin: a breakdown product of hemoglobin released when red cells are destroyed; 
it is made by the liver. Some forms of this bile pigment are highly toxic, and 
may stain and injure brain tissue (kernicterus), causing death. 



T-4 

bradycardia: slowness of the heart beat. 

cannulation: the insertion of a tube. 

carcinogenicity; the tendency to produce a malignant new growth made up of epithelial 
cells. . 

carcinoma: a malignant new growth made up of epithelial cells tending to infiltrate 
the surrounding tissues and transfer the disease. 

cardiac pacemaker, artificial: a device designed to stimulate, by electrical impulses, 
contraction of the heart muscle at a certain rate; used in absence of normal 
function of the sino- atrial node. 

cardiomegaly: enlargement of the heart. 

cataract: an opacity of the crystalline lens of the eye. 

catecholamine: one of a group of similar compounds having a mimicking effect of 

impulses conveyed by the nerve fibers, the aromatic portion of whose molecule 

is catechol, and the aliphatic portion an amine. Such compounds include dopamine, 

norepinephrine, and epinephrine. 

catheterization (cardiac): passage of a small tube through a vein in the arm, leg or 

neck and into the heart, permitting the securing of blood samples, determination 
of intracardiac pressure, and detection of anomalies. 

caudate nucleus: an elongated, arched gray mass closely related to the lateral ventri- 
cal throughout its entire extent and consisting of a head, body, and tail. 

cheinotherapy: treatment of disease by chemical agents. 

chloramphenicol: antimicrobial alcohol produced by Streptomyces venezuelae. 

chlorothiazide: chemical used as a diuretic and antihypertensive. 

chlorpromazine: a phenothiazine derivative; used as a major tranquilizer. 

cholera: an acute infectious disease caused by Vibrio cholerae and characterized by severe 
diarrhea. 

cholesterol: a glistening white soapy crystalline substance, the most common animal 
sterol, a precursor of a form of vitamin D, and a universal tissue constituent, 
occurring notably in bile, gallstones, brain, blood cells, plasma, egg yolk, and 
seeds . 

cholinergic: stimulated, activated, or transmitted by choline (acetylcholine); a term 
applied to those nerve fibers which liberate acetylcholine at a synapse when a 
nerve impulse passes, i.e., the parasympathetic nerve endings. 

cineangiography: the photographic recording of fluoroscopic images of the blood vessels 
by motion-picture techniques. 



T-5 
cirrhosis: liver disease. 

citrated: containing a salt of citric acid. 

coarctation: a condition of stricture or contraction. 

cochlea: the essential organ of hearing; a spirally wound tube, resembling a snail 

shell, which forms part of the inner ear. Its base lies against the lateral end of 
the internal acoustic meatus. 

Collins' solution: a solution resembling intracellular fluid used in perfusion of tissues 
or organs. 

commissurotomy: surgical incision or disruption of a junction of corresponding ana- 
tomical parts, to increase the size of the orifice. 

congenital: existing at, and usually before, birth; referring to conditions that are 
present at birth. 

conjunctiva: the delicate membrane that lines the eyelids. 

contralateral: pertaining to the opposite side. 

corpus luteum (pi. corpora lutea): a yellow glandular mass in the ovary formed by an 
ovarian follicle that has matured and discharged its ovum; if the ovum has been 
impregnated, the corpus luteum increases in size and persists for several 
months; if impregnation has not taken place, the corpus luteum degenerates. 
The corpus luteum secretes progesterone. 

corticosteroid: any of the steroids produced by the adrenal cortex, including Cortisol, 
corticosterone, aldosterone, etc.; also their synthetic equivalents. 

cortisone: a hormone from the adrenal cortex that is largely inactive in man until con- 
verted to Cortisol; active on carbohydrate metabolism. 

counterimmunoelectrophoresis: a method of combining an applied electric field and 
double diffusion for distinguishing between proteins and other materials. The 
antibody is placed in a central well and the antigen in rectangular wells which are 
parallel to the direction of electrophoresis. 

Cr : a chromium radioactive nuclide, used as a tracer. 

cryophylactic: resistant to very low temperatures. 

cutaneous ureterostomy: the operation of bringing the ureter to the skin through an 
incision in the iliac region. 

cyanosis: a bluish discoloration, applied especially to such discoloration of skin and 
mucous membranes due to excessive concentration of reduced hemoglobin in the 
blood. 

cytopathic: pertaining to or characterized by changes in cells caused by disease. 



T-6 

decarboxylase: any of the lysing (remover) class of enzymes that catalyze the removal 
of carbon dioxide from the carboxyl group of alpha keto acids. 

defibrinated: deprived of fibrin. 

dexamethasone: adrenocortical steroid of the glucogenic type. 

diabetes insipidus: metabolic disorder characterized by the loss of large am.ounts of 
urine, great thirst, voracious appetite, loss of strength, and emaciation. 

dialysate: the material that passes through the membrane in dialysis. 

dialysis: the process of separating solutes by differential passage through a membrane. 

dicoumarin: originally isolated from decaying sweet clover, now produced synthetically; 
used as an anticoagulant. 

diethazine: antiparkinsonism agent, possessing atropine- like action. 

dimethisterone: a synthetic progestin. 

diosgenin: an aglycone of the saponin dioscin; obtained from several species of the 

plant genus Dioscorea; it is a precursor in the synthesis of progesterone and other 
medically useful steroids. 

diphtheria: an acute infectious disease caused by the toxigenic bacillus Corynebacterium 
diphtheriae, affecting various membranes. 

diuresis: increased secretion of urine. 

dopa: an amino acid produced by the oxidation of tyrosine by tyrosinase; it is the pre- 
cursor of dopamine and an intermediate product in the biosynthesis of norepine- 
phrine, epinephrine, and melanin. L-dopa, the naturally occurring form, and 
levodopa, a synthetic form, are used in the treatment of parkinsonism and 
manganese poisoning. 

dopamine: a compound, hydroxytyramine, produced by the decarboxylation of dopa, an 
intermediate product in the synthesis of norepinephrine. 

Down's syndrome: see mongolism. 

ductus arteriosus: arterial duct; a fetal blood vessel connecting the pulmonary artery 
directly to the descending aorta. 

ECG: electrocardiogram. 

E. coli (Escherichia coli): a microorganism that constitutes the greater part of the intestinal 
flora of man and other animals. 

embolus: a clot or other plug brought by the blood from another vessel and forced into 
a smaller one, thus obstructing the circulation. 

encephalitis: inflammation of the brain. 



T-7 

endaural: within the ear. 

endocrinal: relating to internal secretions; hormonal. 

endometriuiTi; the mucous membrane of the uterus, the thickness and structure of 
which vary with the phase of the menstrual cycle, 

endosteal: pertaining to the tissue lining the medullary cavity of a bone. 

enteric: pertaining to the small intestine. 

enzyme: a protein capable of producing or accelerating a change in (often a specific) 
compound. 

epinephrine; an adrenal hormone that stimulates autonomic nerve action; can be syn- 
thesized and used as a heart stimulant. Also called adrenalin. 

erythroblastosis fetalis: disease of fetal and early newborn life. Usually occurs when 
red cells from Rh-positive fetus cross the placenta and provoke immune response 
in Rh-negative mother. Her anti-Rh antibodies then enter the fetus, destroying 
its red cells, and stimulating abnormally high production of immature red cells, 
or erythroblasts . 

erythrocyte: see red blood cell. 

estriol: a naturally occurring, relatively weak human estrogen, being a metabolic 
product of estradiol and estron found in high concentrations in the urine. 

estrogen: any of several steroid hormones produced chiefly by the ovary and responsi- 
ble for promoting estrus (regularly recurrent period of ovulation in mammals) 
and the development and maintenance of feinale secondary sex characteristics. 

ethisterone: a steroid resembling both progesterone and testosterone; used as a 
progestin. 

ethopropazine: a chemical homologue of promethazine; its hydrochloride is used in the 
treatment of parkinsonism. 

ethynylestradiol: synthetic estrogen; contraceptive agent. 

etiology: study of the factors that cause disease and the inethod of their introduction 
to the host. 

eustatian tube: a bony and cartilaginous tube connecting the cavity of the middle ear 

with the nasal passage (nasopharynx) and serving to equalize air pressure on both 
sides of the tympanic membrane. 

exanthem: any eruptive disease or eruptive fever; rash. 

extrapyramidal: a functional rather than anatomical unit comprising the nuclei and 
fibers involved in motor activities. 



T-8 

falciparum maLaria: the most serious form of malaria, caused by Plasmodium falciparum. 

fava bean; a plant species (Vicia fava) of legumes whose beans or pollen contain a com- 
ponent that is capable of causing a condition known as favism in susceptible 
individuals, which is described as acute hemolytic anemia. 

Fe : an iron radionuclide used as a tracer. 

fenestration: the surgical creation of a new opening in the labyrinth of the ear for the 
restoration of hearing in cases of otosclerosis. 

fermentation: chemical changes in organic substrates caused by enzymes, generally 
those of living microorganisms. 

fetuin: low-molecular- weight globulin which makes up most of the total globulin in the 
blood of the fetus and newborn of ungulates. 

fibrillation: a small, local involuntary contraction of muscle, invisible under the skin, 
resulting from spontaneous activation of single muscle cells or fibers. 

fibroblast: a tissue connective cell. 

fistula; an abnormal passage or communication, usually between two internal organs, 
or leading from an internal organ to the surface of the body. 

gamma globulin; that part of the serum of which antibodies are made. The gamma 
globulin is separable into several parts on the basis of molecular weight. Two 
of these parts are designated 7S and 19S. 

gangliolytic; an agent that blocks the transmission of impulses through the nerve-cell 
bodies outside the central nervous system. 

gene: the biologic unit of heredity, self-reproducing and located at a definite position 
(locus) on a particular chromosome. 

geneticist; a person who studies the biological science that deals with heredity, change, 
and similarity between organisms through time. 

glomerular nephritis; inflammation of the renal clusters of blood vessels or nerve 
fibers . 

glutathione: a tripeptide glycine, isolated from animal and plant tissues. 

glycerinated; treated with or preserved in glycerin. 

glycoprotein: a conjugated protein consisting of a compound of protein with a carbohy- 
drate group. Glycoproteins include the mucins, the mucoids, and the 
chondroproteins. 

glycotic: pertaining to the breakdown of sugars into simpler compounds. 

gram-positive; staining procedure for microorganisms with treated crystal violet. 

gravid: pregnant. 



T-9 

group A: a person whose red cells carry the A antigen but not the B belongs to group A. 

group B: a person whose red cells carry the B antigen but not the A belongs to group B. 

group O: a person whose red cells carry neither the A antigen nor the B belongs to 
group O. 

gynecologist: a person skilled in the branch of medicine which treats diseases of the 
genital tract in women. 

haptoglobin: a heterogenous protein with a large percentage of carbohydrate; a group 

of glycoproteins in the alphas-globulin fraction of serum which share the same or 
similar subunits and have a common property of binding free hemoglobin. 

heart block: reduction or complete lack of coordination in the beating of the atria and 
ventricles of the heart. 

hemagglutination: the clumping of red blood cells or corpuscles. 

hemodialysis: the removal of certain elements from the blood by virtue of the differ- 
ence in the rates of their diffusion through membranes. 

hemodynamic: pertaining to the movements involved in the circulation of the blood. 

hemolytic: characterized by the liberation of hemoglobin. Hemolysis consists of the 

separation of the hemoglobin from the red cells and its appearance in the plasma. 

heparin: a complex organic acid found especially in lung and liver tissue and having 
the ability in certain circumstances to prevent the clotting of blood. 

hepatic coma: unconsciousness from which the patient cannot be aroused, during 
severe liver illness. 

hepatitis: inflammation of the liver. 

hepatocyte: a parenchymal liver cell. 

hepatoma: tumor of the liver, a transition stage between adenoma and carcinoma of 
the liver. 

heterograft: a graft of tissue transplanted between animals of different species. Also 
called zenograft. 

heterologous: made up of tissue not normal to the part. 

heterozygote: an individual possessing different alleles (contrasting characters trans- 
mitted by alternative genes) in regard to a given character. 

hirudin: the active principle of the secretion of the buccal glands of leeches; an 
antithrombin. 

histamine: a white crystalline compound, found in plant and animal tissue, formed from 
histadine by the action of putrifactive bacteria. It is a stimulant of gastric secre- 
tion, and is used medically for blood-vessel dilation. A pharmaceutical 



T-10 

preparation is used to reduce sensitivity to allergens and as a diagnostic aid in 
testing gastric acid formation. 

histocompatible: capable of being accepted and remaining functional; said of that rela- 
tionship between the genotypes of the donor host in which a graft generally will 
not be rejected. 

HL-A antigen: histocompatibility antigen which depends upon a gene of the Y 
chromosome. 

homogenate: material subjected to extreme shredding and mixing. 

homograft: a graft of tissue between individuals of the same species but of different 
genotype. Also called allograft. 

homologue: similar organ or part. 

hormone: a chemical produced in the body which has a specific regulatory function on 
the activity of an organ. 

humoral: pertaining to the fluid materials in the body. 

Huntington's chorea: a rare hereditary disease characterized by chronic condition of 
jerky movements that appear to be well coordinated but are performed involun- 
tarily, and mental deterioration terminating in dementia. 

hydrazine: a colorless, gaseous diamine; also any member of a group of its substitu- 
tion derivatives. 

hydrolysate: a compound produced by hydrolysis. 

hydrops: any abnormal accumulation of serous fluid in the tissues or in a body cavity. 

hyperuricemia: excess of uric acid in the urine. 

hypobilirubinemia: abnormal diminution of bile pigment in the blood. 

hyponatremia: deficiency of sodium in the blood. 

hysterotomy: incision of the uterus. 

iatrogenic: resulting from the activity of physicians. 

icterus: yellowing of the skin. It occurs when excessive destruction of red cells leads 
to a backup of their breakdown products in the body. 

icterus gravis: term describing the shrunken, yellow liver of patients who have 
suffered from a form of hepatitis with massive necrosis of the liver. 

IgG antibody: one of the classes of antibodies. 

IgM antibody: one of the classes of antibodies. 

iliac: of the lower portion of the small intestine. 



T-11 

immunization: the formation by an individual of antibody against a particular antigen. 
Once the individual has reacted immunologically to a given antigen, he will 
respond, quickly, with antibody production whenever that antigen is again present. 
This individual thus is immunized, or has developed an immunity, to that 
antigen. 

immunodiffusion: diffusion of antigen and antibody from separate reservoirs to form 
decreasing concentration gradients in hydrophilic gels. 

immunofluorescence: a method of determining the location of antigen (or antibody) in 
tissue by the pattern of fluorescence resulting when the tissue is exposed to 
specific antibody (or antigen) labeled with a fluorochrome. 

immunoglobulin: a protein of animal origin with known antibody activity. 

immunosuppression: the artificial prevention or diminution of the immune response. 

implanted pacemaker: a cardiac pacemaker inserted or grafted into the subcutaneous 
tissue; also called internal pacemaker. 

incus: the middle of the three ossicles of the ear, which with the stapes and malleus, 
serves to conduct vibrations from the tympanic membrane to the inner ear; also 
called anvil. 

infantile eczema: hypersensitive inherited dermatitis in infants. 

infarction; producing an area of coagulation necrosis in a tissue resulting from the 
obstruction of the local circulation by a thrombus or embolus. 

inguinal: of the groin; the junctural area between the abdomen and thigh. 

intima: general term denoting an innermost structure. 

intrauterine transfusion: a transfusion of red cells into an erythroblastic fetus, usually 
through a thin tube penetrating the mother's abdominal wall, uterus, and into the 
fetal abdominal cavity. 

in utero: in the uterus, e. g., a fetus. 

in vitro: in glass, i.e., in a test tube or laboratory vessel rather than in a living body 
(in vivo) . 

I. P.: intraperitoneally (occurring within the walls of a cavity). 

ischemic: having a deficiency of the blood supply. 

isoantibody: an antibody produced by one individual that reacts with antigens (isoanti- 
gens) of another individual of the same species. 

isoantigen: an antigen that exists in alternate forms in the species and thus can evoke 
an immune response in a member of that species lacking that form of antigen. 

isoimmunization: development of antibodies against an antigen derived from a genet- 
ically dissimilar individual of the same species. 



T-12 

isoprecipitin: an antibody that is active against antigens of animals of the same species 
(but of dissimilar genetic makeup) as the animal in which it is formed. 

I. v.: intravenously. 

jaundice: yellowing of the skin. It occurs when excessive destruction of red cells leads 
to a backup of their breakdown products in the body. 

jejunum: that portion of the small intestine which extends from the duodenum to the 
ileum. 

kaliuresis: an excretion of potassium in the urine. 

kernicterus: condition with severe neural symptoms associated with high levels of 
bilirubin. 

labyrinthine: pertaining to the system of canals constituting the inner ear. 

labyrinthitis: inflammation of the intercommunicating cavities or canals of the inner 
ea r . 

Laennec's cirrhosis: disease of the liver closely associated with chronic excessive 
alcohol ingestion. 

leucocyte: white blood cell or corpuscle. 

leukemia: a progressive, malignant disease of blood-forming organs, characterized 
by distorted proliferation and development of leukocytes and their precursors 
in the blood and bone marrow. 

ligating: tying or binding with thread or cord. 

Locke's solution: a solution of sodium chloride, calcium chloride, potassium chloride, 
sodium bicarbonate, and dextrose; used in physiological experiments to keep the 
mammalian heart beating. 

lymphoblastic leukemia: leukemia assocated with hyperplasia and overactivity of the 
lymphoid tissue. 

lymphocyte: a white blood cell that plays a role in antibody production. 

malleus: the largest of the auditory ossicles, and the one attached to the tympanic 

membrane; its club-shaped head articulates with the incus; also called hammer. 

mastoid: the mastoid (breast shaped) process of the temporal bone. 

mastoiditis: inflammation of the mastoid antrum and cells. 

meatus: a general term for an opening or passageway in the body. 

mendelian: in genetics, the separate and distinct traits that are exhibited by an animal 
or plant, and are dependent on the genetic constitution of the organism; they may 
be recessive or dominant. 



T-13 

Meniere's disease: deafness, noise in the ear, and vertigo resulting from a disease 
of the labyrinth. 

meningitis: inflammation of the three membranes that envelop the brain and spinal 
cord. 

mercurial: a preparation of mercury. 

mestranol: an estrogenic agent, used in combination with various progestogens as an 
oral contraceptive. 

metastatic: transferal of disease from one organ or part to another not directly con- 
nected with it. 

metastatic cancer: transferal of cancer from one organ or part to another not directly 
connected with it. Cancer is a cellular tiimor, which cells exhibit properties of 
invasion and metastasis. 

methylprednisolone: a chemical used as a glucocorticoid. 

mitral: pertaining to the mitral or bicuspid valve. 

mold: a microfungus having a well-marked mycelium or spore mass, especially an 

economically important decomposer growing on dead organic material, as food. 

mongolism: a genetic abnormality in which the genetic material of a chromosome (21) 
is triplicated instead of duplicated. 

monocytic: such as a microsized leukocyte which incorporates foreign matter. 

monozygotic: pertaining to or derived from one egg (zygote) as identical twins, 

mosaicism: in genetics, the presence in an individual of two or more cell lines that are 
karyotypically or genotypically distinct and are derived from a single zygote. 

murine: pertaining to or affecting rats. 

muscarinic: producing a direct stimulation of smooth muscle; pertaining to the deadly 
alkaloid. 

mycology: the study of fungi. 

myelinization: the act of furnishing with or taking on myelin (lipid substance forming 
a sheath around certain nerve fibers). 

myocardium: the middle and thickest layer of the heart wall, composed of the cardiac 
muscle. 

myoma: a tumor made up of muscular elements. 

NAD: nicotinamide-adenine dinucleotide. 

natriuresis: the excretion of abnormal amounts of sodium in the urine. 



T-14 

neonate: newLy born infant. 

neoplasm: any new and abnormal growth. 

nephrectomy: excision of a kidney. 

nephrogenic: forming kidney tissue. 

neurotropism: the quality of having a special affinity for nervous tissue. 

norepinephrine: a hormone secreted by neurons which acts as a transmitter substance 
of the peripheral sympathetic nerve endings and probably of certain synapses in 
the central nervous system. It is also secreted by the adrenal medulla in 
response to splanchnic stimulation, stored in the chromaffin granules, and being 
released predominantly in response to hypotension. A synthetic compound is 
used as a sympathomimetic vasiopressor. 

norethindrone: similar in action to progesterone and used as a progestational agent; 
also used in combination with mestranol as an oral contraceptive. 

norethynodrel: used alone as a gestational agent and, in combination with mestranol, 
as an oral contraceptive. 

nystagmus: an involuntary rapid movement of the eyeball, which may be horizontal, 
vertical, rotatory, or mixed. Barany's symptom: there is no nystagmus if the 
labyrinth is diseased. 

occluding: tightening. 

ophthalmological: pertaining to the study of eyes. 

oral contraceptive: a hormonal compound taken by mouth in order to block ovulation 
and prevent the occurrence of pregnancy. 

orthostatic: pertaining to or caused by standing erect. 

osseous: of the nature or quality of the bone; bony. 

ossicular: pertaining to the auditory bones. 

otic: pertaining to the ear. 

otitis: inflammation of the ear, which may be marked by pain, fever, abnormalities of 
hearing, deafness, and vertigo. 

otologist: physician who specializes in that branch of medicine which deals with the 
ear, its anatomy, physiology, and pathology. 

otosclerosis: the formation of spongy bone in the capsule of the labyrinth of the ear. 

oval window: an oval opening in the inner wall of the middle ear, which is closed by 
the base of the stapes; also called fenestra vestibuli. 



T-15 

oxygenator: a device which mechanically adds air to venous blood which has been 

removed from the body. It is used in combination with one or more pumps for 
maintaining circulation during open heart surgery, and assisting in circulation 
in general. 

palliative: affording relief but not cure. 

parasympathetic; of or pertaining to that division of the autonomic nervous system 
made up of the ocular, bulbar, and sacral divisions. 

parenteral: not through the alimentary canal but rather by injection through some 
other route. 

parkinsonism: a group of neurological disorders characterized by abnormally decreased 
mobility, tremor, and muscular rigidity. 

pathology: the science of disease and its causes. Pathologists conduct autopsies and 
render diagnoses on the basis of tests and analysis of specimens removed from 
patients. In American hospitals, a pathologist often runs the blood bank. 

perfusion: a liquid poured over or through an organ or tissue; or the injection of fluid 
into an artery in order to reach another tissue. 

peristaltic: having a wormlike movement. 

phenothiazine: a greenish, tasteless compound, prepared by fusing diphenylamine 

with sulfur; used as a veterinary antihelmintic. Also used to denote a group of 
major tranquilizers (e.g., chloropromazine) resembling phenothiazine in 
molecular structure. 

phenylalanine: a naturally occurring amino acid, discovered in 1879 by Schulze; essen- 
tial for optimal growth in infants and for nitrogen equilibration in human adults. 

phenylketonuria: an inborn error of metabolism due to the deficiency or defect in an 

enzyme that should catalyze the conversion of phenylalanine to tyrosine. Abbre- 
viated PKU, 

placebo: an inactive substance or preparation given to satisfy the patient's symbolic 
need for drug therapy, and used in controlled studies to determine the efficacy 
of medicinal substances. 

placenta: tissue structure at the fetus' point of attachment to the uterine wall. It is 

richly endowed with blood vessels. Maternal and fetal circulations are separated 
by a very thin membrane, through which nourishment passes into the fetus. 

pneumococcus: an individual organism of the species Diplococcus pneumoniae. 

pneumothorax: an accumulation of air or gas in the pleural space, which may occur 
spontaneously or as a result of trauma, cr be deliberately induced. 

poliomyelitis: an acute viral disease, occurring sporadically and in epidemics, and 

characterized by fever, sore throat, headache, and vomiting, often with stiffness 
of the neck and back; paralysis, muscular atrophy, and often deformity resulting 
in severe cases. Polio. 



T-16 

poliovirus: producing poliomyelitis; epidemics caused by poliovirus are now largely 
controlled by vaccines. 

polycystic: having many cysts. 

polyuria: the passage of a large volume of urine in a given period, characteristic of 
diabetes . 

potentiating agent: that which enhances another agent so that the combined effect is 
greater than the sum of the effects of each used alone. 

primary syphilis: the primary lesion usually appears 10 to 40 days after infection by 
the spirochete Treponema pallidum, and can be identified by darkfield microscopy. 

progestational: that phase of the menstrual cycle just before menstruation, when the 
corpus luteum is active and the endometrium secreting. Also denoting a class 
of pharmaceutical preparations that have effects similar to those of progesterone; 
used in such disorders as dysfunctional uterine bleeding and recurrent abortion. 

progesterone: the hormone produced by the corpora lutea, adrenal cortex, and pla- 
centa whose function is to prepare the uterus for the reception and development 
of the fertilized ovum by inducing secretion in the proliferated glands. 

progestin: the name originally given in 1930 to the crude hormone of the corpora lutea. 
It is now ioslated in pure form and known as progesterone. The name progestin 
is used for certain synthetic or natural progestational agents. 

promethazine: chemical used as an antihistaminic, and also as an antiemetic and 
tranquilizer. 

prophylaxis: the prevention of disease; preventative treatment. 

prostatic: pertaining to the gland in the male which surrounds the neck of the bladder 
and uretha. 

prosthesis: an artifical substitute for a missing body part. 

pruritus: itching; also, the name of various conditions characterized by itching. 

psychosis: a general term for any major mental disorder of organic and/or emotional 
origin characterized by derangement of the personality and loss of contact with 
reality, often with delusions, hallucinations, or illusions. 

psychotropic: exerting an effect upon the mind; capable of modifying mental activity; 
usually applied to drugs that affect the mental state. 

pulmonary atresia: congenital severe narrowing of the opening between the pulmonary 
artery and the right ventricle. 

pulmonary stenosis: narrowing of the opening between the pulmonary artery and the 
right ventricle. 

p-wave: pressure wave. 



T-17 

pyloric stenosis: narrowing of structure of the pylorus (opening of the stomach to the 
duodenum). 

pyocyanin: a blue-green antibiotic pigment. 

pyridoxine: one of the forms of vitamin B/ . 

QRC: deflections of an ECG tracing that represent ventricular activity of the heart. 

quinone: any benzene derivative in which two hydrogen atoms are replaced by two 
oxygen atoms. 

radioimmunoassay: determination of antigen or antibody concentration by means of a 
radioactive-labeled substance that reacts with the substance under test. 

radiopacity: the property of not permitting the passage of radiant energy, such as 
X- rays. 

rauwolfia alkaloids: physiologically active nitrogen-containing organic bases derived 
from the plant genus Rauwolfia , especially reserpine. 

red blood cells: dish-shaped cells carrying hemoglobin which gives them their reddish 
color. Red cells carry oxygen from lungs to all body tissues. Red cell covering, 
or membrane, carries A, B, Rh, and other blood factors, or antigens. 

reductase: any reducing enzyme. 

remission: a diminution or abatement of the symptoms of a disease; also the period 
during which such diminution occurs. 

renal: pertaining to the kidney. 

reserpine: an alkaloid isolated from the root ol Rauwolfia serpentina; used as a tran- 
quilizer and also as an antihypertensive. 

reticulocytosis: an increase in the number of young red blood cells showing a particular 
network when stained. 

Rh hemolytic disease: disease of fetal and early newborn life. It occurs when red cells 
from an Rh-positive fetus cross the placenta and provoke an immune response 
in an Rh-negative mother. Her anti-Rh antibodies then enter the fetus, destroy- 
ing its red cells, and stimulating abnormally high production of immature cells, 
or erythroblasts. 

Rh-negative: a person whose red cells do not carry the Rh factor is Rh-negative. 

Rh-positive: a person whose red cells carry the Rh factor is Rh-positive. 

Rh vaccine: potent anti-Rh antibody, in the form of the 7S fraction of gamma globulin. 
The vaccine is administered to a woman unsensitized to the Rh factor when she 
delivers a baby in order to prevent her from developing an immunity to the Rh 
factor that could cause sickness or death in the next Rh-positive baby she 
conceives. 



T-18 
RhoGAM: a commercial company's registered trade name for Rh vaccine. 

round window: a round opening in the inner wall of the middle ear below and a little 

behind the oval window; it is covered by the secondary tympanic membrane; also 
called fenestra cochlea. 

rubella: German measles; a mild viral infection characterized by a pink discrete and 
confluent macular (spotty) rash. 

rubella syndrome: due to intrauterine rubella infection, characterized most commonly 
by cataracts, heart anomalies, deafness, and mental retardation. 

saluretic: pertaining to the excretion of sodium and chloride ions in the urine. 

saphenous vein: either of the two chief superficial veins of the leg. 

schizophrenia: any of a group of severe emotional disorders characterized by misin- 
terpretation and retreat from reality, delusions, hallucinations, ambivalence, 
inappropriative effects, and withdrawn, bizarre, or regressive behavior. 

sclerosing: undergoing hardening, especially of a part from inflammation and disease. 

sensitization: see immunization. 

serum: the clear, liquid part of blood which reraains after the red cells and clotting 
elements have been removed. The clear liquid which separates from a clot. 

serum hepatitis: an acute viral illness, formerly considered to be transmitted only by 
nonalimentary-canal means (ex. , contaminated needles and the administration 
of blood), but now known to be transmitted by oral ingestion of contaminated 
material. 

7S: a part of the gamma globulin in which antibodies may exist. The 7S gamma globulin 
molecule is relatively small, and will pass through the placenta from mother to 
fetus. 

SH: sulfhydryl. 

sickel cell anemia: a hereditary, genetically determined hemolytic anemia, occurring 
almost exclusively in Negroes, characterized by arthralgia, acute attacks of 
abdominal pain, ulcerations, sickle-shaped erythrocytes in the blood, and the 
presence of homozygous S hemoglobin in red blood cells. 

sitosterol: a generic term for a group of closely related natural plant sterols, the 
individual compounds being designated by Greek letters. 

6 -mercaptopurine: a chemical used for inhibiting or preventing maturation and prolif- 
eration of malignant cells. 

somatherapeutic: curing the ills of the body. 

speculum: an instrument for exposing the interior of a passage or cavity of the body. 



T-19 

spherocytosis: the presence of spherocytes (small, globular, completely hemoglobinated 
erythrocytes without the usual central pallor) in the blood. 

spina bifida: developmental anomaly; defective closure of the bony encasement of the 
spinal cord, through which the cord and meninges may or may not protrude. 

stapes: the innermost of the auditory ossicles (three bones of the inner ear)-, shaped 
somewhat like a stirrup; it articulates by its head with the incus (middle of the 
three ossicles), and its base is inserted into the oval window; also called stirrup. 

steroid: any of a numerous naturally occurring, fat-soluble organic compounds having 
a 17-carbon-atom ring as a basis. Some of the substances included in this group 
are progesterone, sterols (such as cholesterol), digitalis compounds, and pre- 
cursors of certain vitamins. 

sterol: any of a group of predominantly unsaturated solid alcohols of the steroid group, 
such as cholesterol and ergosterol, occurring in the fatty tissues of plants and 
animals . 

Stokes-Adams syndrome: a condition caused by heart block and characterized by 
sudden attacks of unconsciousness, with or without convulsions. Also called 
Adams-Stokes disease. 

streptomycin: a bactericidal antibiotic which is produced by a soil fungus, and effec- 
tive against certain bacteria but used chiefly for the treatment of tuberculosis. 

striatum: one of the components of the basal ganglia; a subcortical mass of gray and 
white substances in front of and lateral to the thalamus in each cerebral 
hemisphere. 

strophanthidin: an aglycone obtained by hydrolysis of glycosides from the plant 
Strophanthus kombe. 

sulfanilamide: a potent antibacterial compound; has been replaced by more effective 
and less toxic derivatives, in usage. 

sulphobromophthalein: a chemical used as a hepatic-function determinant. 

supranuclear palsy (pseudobulbar paralysis): spastic weakness of the muscles inner- 
vated by cranial nerves, i.e., the muscles of the face, pharynx, and tongue, due 
to bilateral lesions of the cortospinal tract; it is often accompanied by uncon- 
trolled weeping or laughing. 

sympathetic: the thoracic and lumbar portion of the autonomic nervous system. 

syndrome: a set of symptoms that occur together. 

synthesis: the artificial building up of a chemical compound, by the union of its ele- 
ments or from other suitable starting materials. 

tachycardia: excessive rapidity in action of the heart. 

tamponade: pathologic compression of a part. 



T-20 

temporal bone: one of two irregular bones forming part of the lateral surfaces and 
base of the skull, and containing the organs of hearing. 

teratoblastoma: a neoplasm containing embryonic elements and differing from a tera- 
toma in that its tissue does not represent all the germinal layers. 

terramycin: preparations of an antibiotic substance produced by Streptomyces rimosus , 
which are effective against a wide range of microorganisms. 

tetralogy of Fallot: a combination of four cardiac defects. 

thebesian vein: the smallest cardiac veins which arise in the muscular walls and drain 
independently into the cavities of the heart, and most readily seen in the atria. 

Thiersch graft: a very thin skin graft in which long, thin strips of skin, consisting of 
the epidermis and other tissues, are used. Also called Ollier-Thiersch graft. 

thoracotomy: surgical incision of the wall of the chest. 

thromboembolism: obstruction of a blood vessel with an aggregation of blood emboli 
carried by the blood stream from the site of origin to plug another vessel. 

thrombose; an obstructing clot at the point of formation. 

thrombosis: the formation, development, or presence of a thrombus. 

thrombus: an aggregation of blood factors, primarily platelets and fibrin with entrap- 
ment of cellular elements, frequently causing vascular obstruction at the point 
of formation. 

titer: the quantity of a substance required to produce a reaction with a given volume of 
another substance, or the amount of one substance required to correspond with 
a given amount of another substance. 

torsion systonia: a rare, chronic, hereditary disease marked by involuntary, irregular, 
muscle contractions and relaxations in the trunk and extremity areas, 

transferrin: serum beta-globulin (intermediate to alpha- and gamma- globulins) that 
binds and transports iron. 

transtympanic: across the tympanic membrane or the cavity of the middle ear. 

tricuspid atresia: congenital absence or closure of tricuspid valve. 

tympanic mucosa (tympanic membrane): a thin membrane closing externally the cavity 
of the middle ear like the head of a drum, and in humans, being deeply located 
at the bottom of the external auditory meatus. Also called membrana tympani. 

Type 1 poliovirus: one of three serotypes of poliovirus. 

Tyrode's solution: a modified Locke's solution containing magnesium, used especially 
for perfusing the intestine. 



T-21 

tyrosine: a crystallizable amino acid, found in most proteins and synthesized meta- 
bolicaLly from phenyLalanine. 

tyrothricin: an antibiotic substance isolated from the soil bacillus Bacillus brevis. 

uremia: the retention of excessive by-products of protein metabolism in the blood, and 
the toxic condition produced. 

ureter: the fibromuscular tube which conveys urine from the kidney to the bladder. 

urochrome: a yellow, amorphous pigment of the urine which gives urine its yellow 
color. 

vaccinia: a viral disease of cattle. 

vaccine: a substance introduced into the body to prevent disease immunologically. 

valvuloplasty: plastic (surgery) operation on a valve. 

vertigo: an illusion of inovement; may result from diseases of the inner ear or may be 
due to disturbances of the central nervous system. 

viremic: pertaining to the presence of viruses in the blood. 

virus (wild and attenuated): one of a group of minute infectious agents. 

Vitallium: trademark for a cobalt-chromium alloy used in surgical equipment. 

X-chromosome: the femal sex chromosome, being the differential sex chromosome 
carried by half the male gametes and all female gametes in humans. 



ft U.S. GOVERNMENT PRINTING OFFICE : 1976 O-207-613 



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